Isolation, identification and characterization of tmst2, a novel member of the TNF-receptor supergene family

ABSTRACT

Novel TNF reccptor polypeptides are disclosed, along with polynucleotides encoding the polypeptides and uses thereof.

RELATED APPLICATIONS

This patent application claims priority from U.S. provisional patentapplication No. 60/143,063 filed Jul. 9, 1999.

FIELD OF THE INVENTION

The invention is in the field of recombinant genetics. In particular,the present invention relates to a novel transmembrane decoy-receptor,tmst2, and its secreted splice variant, belonging to the TNF-receptorsuper genie family and nucleic acid molecules encoding same. Theinvention also relates to vectors, host cells, antibodies andrecombinant methods for producing both the membrane associated and thesoluble forms of the receptor polypeptides. The invention also relatesto the use of the recombinant tmst2 receptor polypeptide to identifyputative binding proteins. In addition, methods and reagents areprovided for the diagnosis of diseases associated with or resulting fromabnormal tmst2 and/or abnormal expression of its putative ligand, andmethods and pharmaceutical composition(s) for the treatment,amelioration and/or treatment of diseases associated with abnormal tmst2or abnormal expression of tmst2 and/or its ligand. The invention alsodiscloses pharmaceutical compositions for use in the treatment of thesediseases.

BACKGROUND OF THE INVENTION

Technical advances in the identification, cloning, expression andmanipulation of nucleic acid molecules have greatly accelerated thediscovery of novel therapeutics based upon deciphering the human genome.Rapid nucleic acid sequencing techniques can now generate sequenceinformation at unprecedented rates, and coupled with computationalanalyses, allow the assembly of overlapping sequences into the entiregenome and the identification of polypeptide-encoding regions.Comparison of a predicted amino acid sequence against a databasecompilation of known amino acid sequences can allow one to determine theextent of homology to previously identified sequences and/or structurelandmarks. Cloning and expression of a polypeptide-encoding region of anucleic acid molecule provides a polypeptide product for structural andfunctional analysis. Manipulation of a nucleic acid molecule(s) andencoded polypeptide(s) to give variants and derivatives thereof mayconfer advantageous properties on a product for use as a therapeutic.

However, in spite of the significant technical advances in genomeresearch over the past decade, the potential for development of noveltherapeutics based on the human genome is still largely unrealized.While a number of genes encoding potentially beneficial proteintherapeutics, or those encoding polypeptides which may act as “targets”for therapeutic molecules, have been identified using recombinant DNAtechnology, the structure and function of a vast number of genes in thegenome of mammals are yet unknown.

Using the above mentioned recombinant DNA technology, we have recentlyidentified a new member of the tumor necrosis factor (TNF)-receptorsupergene family, hereinafter referred to as “tmst2”, and secretedsplice variant of tmst2-receptor polypeptide, hereinafter referred to astmst2 which may elicit its effects by binding a member of the TNF-familyof ligands.

Identification and Characterization of TNF-Family of Ligands andReceptors

Tumor necrosis factor (TNF) was first identified in the serum of miceand rabbits which had been infected with bacillus of Calmette andGuerin(BCG) and which had been injected with endotoxin. TNF activity inthe serum of these animals was recognized on the basis of its cytotoxicand anti-tumor activities. This TNF activity, referred to as TNF-α, isproduced particularly by activated monocytes and macrophages, and hasbeen implicated in normal growth processes as well as in a variety ofdiseases.

Following the discovery of TNF-α, independent research led to theidentification of another cytokine associated with inflammatoryresponses lymphotoxin-α (LT-α) which was shown to be producedexclusively by lymphocytes. LT-α was subsequently shown to be 30%homologous with TNF-α, and was renamed TNF-β. It is now clear that TNF-αand TNF-β are members of a gene family that includes yet another membertermed LT-β (Browning et al., Cell 72:847-856 (1993)). The three genesare tightly linked within the MHC complex and show similar organization.Moreover, the biologically active forms of TNF-α and TNF-β arehomotrimers and share many of the same biological activities includingcompeting for the same cell-surface receptors (Agarwal et al., Nature318:665-667 (1985)). Two distinct but structurally homologous receptorshave been identified, and each has been shown to bind both the ligandsand mediate their effects.

However, it has been recognized that TNFs are only representativemembers of the rapidly expanding supergene family that includes TNF-α,TNF-β/lymphotoxin-α (LT-α), lynphotoxin-β (LT-β), FasL, CD40L, CD30L,CD27L, 4-IBBL, and TNF-related apoptosis-inducing ligand (TRAIL), RANKL,GITRL and TNF-2. The distinctive but overlapping cellular responsesinduced by members of the TNF family of ligands following theirinteraction(s) with their cognate cell-surface receptors result inclearly defined developmental and regulatory changes in cells of thelymphoid, hematopoietic, and other lineages. For example, TNF family ofligands are involved in growth regulation and differentiation of cellswhich are involved in inflammation, immune processes and hematopoiesis(Bayert, R. and Fiers, W., Tumor Necrosis Factor and Lymphokines in:Cytokines eds. Anthony Mire-Sluis and Robin Thorpe, Academic Press SanDiego Calif. (1998)). TNF family of ligands activate the immune defensesagainst parasites, and acts directly and/or indirectly as a mediator inimmune reactions and inflammatory processes. However, administration ofTNF and/or other members of the TNF family can also be accompanied byharmful phenomena such as shock and tissue damage (Bayert, R. and Fiers,W., supra). The main physiological role of TNF family of ligands islikely the activation of first-line reaction of an organism tomicrobial, parasitic, viral, or to mechanical stress and cancer. Forexample, TNF-related apoptosis-inducing ligand (TRAIL) has beendemonstrated to induce apoptosis of a number of different types ofcancer cells as well as virally infected cells.

Furthermore, a number of observations have also led to the conclusionthat TNF family of ligands are also involved in a variety ofpathological conditions including cachexia, toxic shock syndrome,inflammatory diseases such as rheumatoid and osteoarthritis, in deathresulting from graft-versus- host reaction (GVHR)(Bayert, R. and Fiers,W., supra), rapid necrosis of tumors, apoptosis, immunostimulation andresistance to parasites and viruses.

Like other cytokines, the TNF family of ligands binds to specific cellsurface receptors. Based upon sequence similarities, the TNF receptorsbelong to a receptor gene super-family that includes the low-affinitynerve growth factor (NGF) receptor, the FAS antigen, the humanB-lynphocyte activation molecule CD40, CD27, 4-1BB, PV-T2, CD30, TNFR-RP, TRAIL-R, PV-A53R, RANK, GITR and OX40 antigen found on activated Tcells (Smith et al., Cell, 76: 959-62 (1994): Baker and Reddy, Oncogene,12: 1-9 (1996)). Sequence similarities between any two family membersmay exist throughout the molecule, or be confined to the extracellularor intracellular domain. The intracellular domain of some of thereceptors contains a so-called death domain (DD), which mediatesligand-induced programmed cell death (apoptosis). The pathways employedto induce death differ among death domains of individual TNF receptors.For example, the FAS antigen DD signals through FADD, RIP and caspase-8;the TNFR-1 signals through FADD, TRADD and caspase-8; and the deathdomain of the TRAIL-receptor DR4 induces apoptosis without interactingwith any of the above adapter molecules. The sequence diversity amongextracellular domains of the TNF receptor family is reflected in theirbinding specificities: some bind TNF, others do not.

In addition to the membrane associated receptor molecules describedabove, a number the receptors belonging to the TNF-receptor supergenefamily exist as soluble binding proteins. Many of the soluble forms ofthe transmembrane receptors were subsequently identified as containingonly the extracellular ligand binding domain(s) of the receptors. Forexample, a soluble form of TNF receptor has been found in urine andserum (See U.S. Pat. No.: 5,843,789 and Nophar et al. EMBO J., 9(10):3269-78 (1990)), and have been shown to arise by proteolytic cleavage ofcell surface TNF-receptors (Porteu et al., J. Biol. Chem., 266: 18846-53(1991)). These soluble forms of receptor molecules have been implicatedin the modulation of TNF activity by not only interfering with TNFbinding to its receptor, but also by stabilizing the structure andpreserving its activity, thus prolonging some of its effects (Aderka etal, Cytokine & Growth Factor Reviews, 7(3):231-240 (1996)).

The activity of TNF family of ligands are tightly regulated at thelevels of secretion and receptor expression. Additional regulatorymechanisms are provided by action of specific inhibitory proteinspresent on cell surface and in biological fluids. While some of theseinhibitory proteins have been identified as soluble forms of receptormolecules, the identity of many of these cytokine regulatory proteinsare as yet unknown. However, abnormalities in the production of thesesubstances might contribute to the pathophysiology of a variety ofdiseases including immune and neoplastic diseases. Besides their role inregulating cytokine activity in vivo, these regulatory molecules holdsignificant potential for therapeutic use as very specificinhibitors/anti-cytokine agents, and as indicators in diagnosis andassessment of immune function and growth parameters in a variety ofautoimmune and malignant diseases (Fernandez-Botran, FASEB J., 5:2567-74 (1991)).

Accordingly, the invention is directed to novel nucleic acid moleculesencoding TNF-receptor(s) related molecule(s) that regulate the activityof TNF family of ligands, and to polypeptides encoded by the nucleicacids, as well as their use as diagnostic and/or therapeutic moleculesof diseases.

SUMMARY OF THE INVENTION

The present invention relates to novel tmst2-receptor nucleic acidmolecules and encoded polypeptides.

The invention provides for an isolated nucleic acid molecule comprisinga nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence as set forth in SEQ ID NO: 7 or 9;

(b) a nucleotide sequence encoding the polypeptide as set forth in SEQID NO: 8 or 10;

(c) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of (a) or (b), wherein theencoded polypeptide has an activity of the polypeptide as set forth inSEQ ID NO: 8 or 10; and

(d) a nucleotide sequence complementary to any of (a)-(c).

The invention also provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide that is at least about70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to thepolypeptide as set forth in SEQ ID NO: 8 or 10 as determined using acomputer program selected from the group consisting of GAP, BLASTP,BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Watermanalgorithm, wherein the polypeptide has an activity of the polypeptide asset forth in SEQ ID NO: 8 or 10;

(b) a nucleotide sequence encoding an allelic variant or splice variantof the nucleotide sequence as set forth in SEQ ID NO: 7 or 9, whereinthe encoded polypeptide has an activity of the polypeptide as set forthin SEQ ID NO: 8 or 10;

(c) a nucleotide sequence of SEQ ID NO: 7 or 9, (a), or (b) encoding apolypeptide fragment of at least about 25 amino acid residues, whereinthe polypeptide has an activity of the polypeptide as set forth in SEQID NO: 8 or 10;

(d) a nucleotide sequence encoding a polypeptide that has a substitutionand/or deletion of 1 to 198 amino acid residues as set forth in any ofSEQ ID NOS: 7-8 wherein the encoded polypeptide has an activity of thepolypeptide as set forth in SEQ ID NO: 8 or 10;

(d) a nucleotide sequence encoding a polypeptide that has a substitutionand/or deletion of 1 to 100 amino acid residues as set forth in any ofSEQ ID NOS: 9-10 wherein the encoded polypeptide has an activity of thepolypeptide as set forth in SEQ ID NO: 8 or 10;

(f) a nucleotide sequence of SEQ ID NO: 7 or 9, or (a)-(f) comprising afragment of at least about 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(f), wherein thepolypeptide has an activity of the polypeptide as set forth in SEQ IDNO: 8 or 10; and

(h) a nucleotide sequence complementary to any of (a)-(f).

The invention further provides for an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of.

(a) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 8 or 10 with at least one conservative amino acid substitution,wherein the polypeptide has an activity of the polypeptide as set forthin SEQ ID NO: 8 or 10;

(b) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 8 or 10 with at least one amino acid insertion, wherein thepolypeptide has an activity of the polypeptide as set forth in SEQ IDNO: 8 or 10;

(c) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 8 or 10 with at least one amino acid deletion, wherein thepolypeptide has an activity of the polypeptide as set forth in SEQ IDNO: 8 or 10;

(d) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 8 or 10 which has a C- and/or N-terminal truncation, wherein thepolypeptide has an activity of the polypeptide as set forth in SEQ IDNO: 8 or 10;

(e) a nucleotide sequence encoding a polypeptide as set forth in SEQ IDNO: 8 or 10 with at least one modification selected from the groupconsisting of amino acid substitutions, amino acid insertions, aminoacid deletions, C-terminal truncation, and N-terminal truncation,wherein the polypeptide has an activity of the polypeptide as set forthin SEQ ID NO: 8 or 10;

(f) a nucleotide sequence of (a)-(e) comprising a fragment of at leastabout 16 nucleotides;

(g) a nucleotide sequence which hybridizes under moderately or highlystringent conditions to the complement of any of (a)-(f), wherein thepolypeptide has an activity of the polypeptide as set forth in SEQ IDNO: 8 or 10; and

(h) a nucleotide sequence complementary to any of (a)-(e).

The invention also provides for an isolated polypeptide comprising theamino acid sequence selected from the group consisting of:

(a) the mature amino acid sequence as set forth in SEQ ID NO: 8 or 10comprising a mature amino terminus at residue(s) 1, and optionallyfurther comprising an amino-terminal methionine;

(b) an amino acid sequence for an ortholog of SEQ ID NO: 8 or 10,wherein the encoded polypeptide has an activity of the polypeptide asset forth in SEQ ID NO: 8 or 10;

(c) an amino acid sequence that is at least about 70, 80, 85, 90, 95,96, 97, 98, 5 or 99 percent identical to the amino acid sequence of SEQID NO: 8 or 10 as determined using a computer program selected from thegroup consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit,and the Smith-Waterman algorithm;, wherein the polypeptide has anactivity of the polypeptide as set forth in SEQ ID NO: 8 or 10;

(d) a fragment of the amino acid sequence set forth in SEQ ID NO: 8 or10 comprising at least about 25 amino acid residues, wherein thepolypeptide has an activity of the polypeptide as set forth in SEQ IDNO: 8 or 10;

(e) an amino acid sequence for an allelic variant or splice variant ofeither the amino acid sequence as set forth in SEQ ID NO: 8 or 10, or atleast one of(a)-(c) wherein the polypeptide has an activity of thepolypeptide as set forth in SEQ ID NO: 8 or 10.

The invention further provides for an isolated polypeptide comprisingthe amino acid sequence selected from the group consisting of:

(a) the amino acid sequence as set forth in SEQ ID NO: 8 or 10 with atleast one conservative amino acid substitution, wherein the polypeptidehas an activity of the polypeptide as set forth in SEQ ID NO: 8 or 10;

(b) the amino acid sequence as set forth in SEQ ID NO: 8 or 10 with atleast one amino acid insertion, wherein the polypeptide has an activityof the polypeptide as set forth in SEQ ID NO: 8 or 10;

(c) the amino acid sequence as set forth in SEQ ID NO: 8 or 10 with atleast one amino acid deletion, wherein the polypeptide has an activityof the polypeptide as set forth in SEQ ID NO: 8 or 10;

(d) the amino acid sequence as set forth in SEQ ID NO: 8 or 10 which hasa C- and/or N-terminal truncation, wherein the polypeptide has anactivity of the polypeptide as set forth in SEQ ID NO: 8 or 10; and

(e) the amino acid sequence as set forth in SEQ ID NO: 8 or 10, with atleast one modification selected from the group consisting of amino acidsubstitutions, amino acid insertions, amino acid deletions, C-terminaltruncation, and N-terminal truncation, wherein the polypeptide has anactivity of the polypeptide as set forth in SEQ ID NO: 8 or 10.

Also provided are fusion polypeptides comprising the amino acidsequences of (a)-(e) above.

The present invention also provides for an expression vector comprisingthe isolated nucleic acid molecules as set forth herein, recombinanthost cells comprising recombinant nucleic acid molecules as set forthherein, and a method of producing a tmst2-receptor polypeptidecomprising culturing the host cells and optionally isolating thepolypeptide so produced.

A transgenic non-human animal comprising a nucleic acid moleculeencoding a tmst2-receptor polypeptide is also encompassed by theinvention. The tmst2-receptor nucleic acid molecules are introduced intothe animal in a manner that allows expression and increased levels ofthe tmst2-receptor polypeptide, which may include increased circulatinglevels. The transgenic non-human animal is preferably a mammal.

Also provided are derivatives of the tmst2-receptor polypeptides of thepresent invention.

Additionally provided are selective binding agents such as antibodiesand peptides capable of specifically binding the tmst2-receptorpolypeptides of the invention. Such antibodies and peptides may beagonistic or antagonistic.

Pharmaceutical compositions comprising the nucleotides, polypeptides, orselective binding agents of the present invention and one or morepharmaceutically acceptable formulation agents are also encompassed bythe invention. The pharmaceutical compositions are used to providetherapeutically effective amounts of the nucleotides or polypeptides ofthe present invention. The invention is also directed to methods ofusing the polypeptides, nucleic acid molecules, and selective bindingagents. The invention also provides for devices to administer atmst2-receptor polypeptide encapsulated in a membrane.

The tmst2-receptor polypeptide(s) of the invention and its biologicallyactive variant(s), analog(s) and fragment(s) maybe used for therapeuticand/or diagnostic purposes to treat, prevent and/or detect conditionsresulting from the abnormal expression of tmst2-receptor polypeptide orfrom the abnormal expression of a putative tmst2-ligand or a member ofthe TNF family of ligands that bind to tmst2-receptor polypeptide causedby overreaction of the host or deficiency of a natural autoregulatorynetwork such as frequently observed in sepsis, cachexia, auto-immuneresponses, inflammatory diseases, viral, bacterial and parasiticdiseases, and cancer.

The invention encompasses diagnosing a pathological condition or asusceptibility to a pathological condition in a subject caused by orresulting from abnormal levels of tmst2-receptor polypeptide comprisingdetermining the presence or amount of expression of the tmst2-receptorpolypeptide in a sample; and comparing the level of said polypeptide ina biological, tissue or cellular sample from either normal subjects orthe subject at an earlier time, wherein susceptibility to a pathologicalcondition is based on the presence or amount of expression of thepolypeptide.

The present invention also provides a method of assaying test moleculesto identify a test molecule which binds to a tmst2-receptor polypeptide.The method comprises contacting a tmst2-receptor polypeptide with a testmolecule and determining the extent of binding of the test molecule tothe polypeptide. The method further comprises determining whether suchtest molecules are agonists or antagonists of a tmst2-receptorpolypeptide. The present invention further provides a method of testingthe impact of molecules on the expression of tmst2-receptor polypeptideor on the activity of tmst2-receptor polypeptide.

Methods of regulating expression and modulating (i.e., increasing ordecreasing) levels of a tmst2-receptor polypeptide are also encompassedby the invention. One method comprises administering to an animal anucleic acid molecule encoding a tmst2-receptor polypeptide. In anothermethod, a nucleic acid molecule comprising elements that regulate ormodulate the expression of a tmst2-receptor polypeptide may beadministered. Examples of these methods include gene therapy, celltherapy, and anti-sense therapy as further described herein.

The tmst2-receptor polypeptide can be used for identifying ligandsthereof. Various forms of “expression cloning” have been used forcloning ligands for receptors. See e.g., Davis et al., Cell 87:1161-1169(1996). These and other tmst2-receptor ligand cloning experiments aredescribed in greater detail herein. Isolation of the tmst2-receptorligand(s) allows for the identification or development of novel agonistsand/or antagonists of the tmst2-receptor signaling pathway. Suchagonists and antagonists include tmst2-receptor ligand(s),anti-tmst2-receptor ligand antibodies and derivatives thereof, smallmolecules, or antisense oligonucleotides, any of which can be used forpotentially treating one or more diseases or disorders, including thoserecited herein.

DETAILED DESCRIPTION OF THE INVENTION

The section headings herein are for organizational purposes only and arenot to be construed as limiting the subject matter described therein.

Definitions:

The term “tmst2 receptor nucleic acid molecule” refers to a nucleic acidmolecule comprising or consisting essentially of or comprising anucleotide sequence as set forth in SEQ ID NO: 7 or 9, comprising orconsisting essentially of a nucleotide sequence encoding the polypeptideas set forth in SEQ ID NO: 8 or 10, or nucleic acid molecules relatedthereto. Related nucleic acid molecules comprise or consist essentiallyof a nucleotide sequence that is about 70 percent identical to thenucleotide sequence as shown in SEQ ID NO: 7 or 9, or comprise orconsist essentially of a nucleotide sequence encoding a polypeptide thatis about 70 percent identical to the polypeptide as set forth in SEQ IDNO: 8 or 10. In preferred embodiments, the nucleotide sequences areabout 75 percent, or about 80 percent, or about 85 percent, or about 90percent, or about 95, 96, 97, 98, or 99 percent identical to thenucleotide sequence as shown in SEQ ID NOS: 7 or 9, or the nucleotidesequences encode a polypeptide that is about 75 percent, or about 80percent, or about 85 percent, or about 90 percent, or about 95, 96, 97,98, or 99 percent identical to the polypeptide sequence as set forth inSEQ ID NOS: 8 or 10. Related nucleic acid molecules also includefragments of the above tmst2 receptor nucleic acid molecules which areat least about 10 contiguous nucleotides, or about 15, or about 20, orabout 25, or about 50, or about 75, or about 100, or greater than about100 contiguous nucleotides.

Related nucleic acid molecules also include fragments of the abovetmst2-receptor nucleic acid molecules which encode a polypeptide of atleast about 25 amino acid residues, or about 50, or about 75, or about100, or greater than about 100 amino acid residues. Related nucleic acidmolecules also include a nucleotide sequence encoding a polypeptidecomprising or consisting essentially of a substitution and/or a deletionof one or more of the 198 amino acid residues set out in SEQ ID NO: 8.Related nucleic acid molecules also include a nucleotide sequenceencoding a polypeptide comprising or consisting essentially of asubstitution and/or a deletion of one or more of the amino acid residuesset out in SEQ ID NO: 10. Related tmst2-receptor nucleic acid moleculesinclude those molecules which comprise nucleotide sequences whichhybridize under moderate or highly stringent conditions as definedherein with any of the above nucleic acid molecules. In preferredembodiments, the related nucleic acid molecules comprise sequences whichhybridize under moderate or highly stringent conditions with thesequence as shown in SEQ ID NO: 7 or 9, or with a molecule encoding apolypeptide, which polypeptide comprises the sequence as shown in SEQ IDNO: 8 or 10, or with a nucleic acid fragment as defined above, or with anucleic acid fragment encoding a polypeptide as defined above. It isalso understood that related nucleic acid molecules include allelic orsplice variants of any of the above nucleic acids, and include sequenceswhich are complementary to any of the above nucleotide sequences.

The term “nucleic acid sequence” or “nucleic acid molecule” refers to aDNA or RNA sequence. The term encompasses molecules formed from any ofthe known base analogs of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinyl-cytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosinie, 2,2-dimethyl-guanine,2-methyladenine, 2-methylguaninie, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguaninic, 5-methylaminomethyluracil,5-metihoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosile,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosinc, 2-thiocytosine, and2,6-diaminopurine.

The term “naturally occurring” or “native” when used in connection withbiological materials such as nucleic acid molecules, polypeptides, hostcells, and the like, refers to materials which are found in nature andare not manipulated by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that (1) has been separated fromat least about50 percent of proteins, lipids, carbohydrates or other materials withwhich it is naturally found when total DNA is isolated from the sourcecells, (2) is not linked to all or a portion of a polynucleotide towhich the “isolated nucleic acid molecule” is linked in nature, (3) isoperably linked to a polynucleotide which it is not linked to in nature,or (4) does not occur in nature as part of a larger polynucleotidesequence. Preferably, the isolated nucleic acid molecule of the presentinvention is substantially free from any other contaminating nucleicacid molecule(s) or other contaminants that are found in its naturalenvironment that would interfere with its use in polypeptide productionor its therapeutic, diagnostic, prophylactic or research use.

The term “allelic variant” refers to one of several possible naturallyoccurring alternate forms of a gene occupying a given locus on achromosome of an organism.

The term “splice variant” refers to nucleic acid molecule, usually RNA,which is generated by alternative processing of introni sequences in anRNA transcript.

The term “expression vector” refers to a vector which is suitable forpropagation in a host cell and contains nucleic acid sequences whichdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in Genetic make-up to the original parent, solong as the selected gene is present.

The term “operably linked” is used herein to refer to an arrangement offlanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Thus, aflanking sequence operably linked to a coding sequence may be capable ofeffecting the replication, transcription and/or translation of thecoding sequence. For example, a coding sequence is operably linked to apromoter when the promoter is capable of directing transcription of thatcoding sequence. A flanking sequence need not be contiguous with thecoding sequence, so long as it functions correctly. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more fonnulationmaterials suitable for accomplishing or enhancing the delivery of thetmst2-receptor-receptor like polypeptide, tmst2-receptor-receptor likenucleic acid molecule or tmst2-receptor-receptor like selective bindingagent as a pharmaceutical composition.

The term “selective binding agent” refers to a molecule or moleculeshaving specificity for an tmst2-receptor-receptor like polypeptide. Asused herein, the terms, “specific” and “specificity” refer to theability of the selective binding agents to bind to humantmst2-receptor-receptor like polypeptides and not to bind to humannon-tmst2-receptor-receptor like polypeptides. It will be appreciated,however, that the selective binding agents may also bind orthologs ofthe polypeptide as set forth in SEQ ID NO: 8 or 10, that is,interspecies versions thereof, such as mouse and rat polypeptides.

The term “transduction” is used to refer to the transfer of genes fromone bacterium to another, usually by a phage. “Transduction” also refersto the acquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, for example, Gralham et al., Virology, 52:456 (1973);Sambrook et al. Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratories (New York, 1989); Davis et al., Basic Methods inMolecular Biology, Elsevier, 1986; and Chu et al., Gene. 13:197 (1981).Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transfonred when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, may be maintained transiently as an episomal element without beingreplicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell.

The term “tmst2-receptor polypeptide” refers to a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 8 or 10, and related polypeptidesdescribed herein. Related polypeptides includes allelic variants, splicevariants, fragments, derivatives, substitution, deletion, and/orinsertion variants, fusion polypeptides, and orthologs.tmst2-receptor-receptor polypeptide(s) may be mature polypeptide(s), asdefined herein, and may or may not have an amino terminal methionineresidue, depending on the method by which they are prepared.

The term “tmst2-receptor polypeptide fragment” refers to a peptide orpolypeptide that comprises less than the full length amino acid sequenceof a tmst2-receptor polypeptide as set forth in SEQ ID NO: 8 or 10. Sucha fragment may arise, for example, from a truncation at the aminoterminus, a truncation at the carboxy terminus, and/or an internaldeletion of the amino acid sequence. Tmst2-receptor fragments may resultfrom alternative RNA splicing or from in vivo protease activity. Fortransmembrane or membrane-bound forms of a tmst2-receptor polypeptide,preferred fragments include soluble forms such as those lacking atransmembrane or membrane-binding domain.

The term “tmst2-receptor polypeptide variants” refers to tmst2-receptorpolypeptides comprising amino acid sequences which contain one or moreamino acid sequence substitutions, deletions (such as internal deletionsand/or tmst2-receptor fragments), and/or additions (Such as internaladditions and/or tmst2-receptor like fusion polypeptides) as compared tothe tmst2-receptor polypeptide amino acid sequence set forth in SEQ IDNO:8 or 10. Variants may be naturally occurring (e.g., tmst2-receptorallelic variants, tmst2-receptor orthologs, and tmst2-receptor splicevariants) or artificially constructed using recombinant DNA technology.Such tmst2-receptor polypeptide variants may be prepared from thecorresponding nucleic acid molecules encoding said variants, which havea DNA sequence that varies accordingly from the DNA sequences for wildtype tmst2-receptor polypeptides as set forth in SEQ ID NOS: 7 or 9. Inpreferred embodiments, the variants have from 1 to 3, or from 1 to 5, orfrom 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acidsubstitutions, insertions, additions and/or deletions, wherein thesubstitutions may be conservative, or non-conservative, or anycombination thereof.

The term “tmst2-receptor like polypeptide fragment” refers to apolypeptide that comprises a truncation at the amino terminus (with orwithout a leader sequence) and/or a truncation at the carboxy terminusof the polypeptide as set forth in SEQ ID NO: 8 or 10, tmst2-receptorpolypeptide allelic variants, tmst2-receptor polypeptide orthologs,tmst2-receptor polypeptide splice variants and/or an tmst2-receptorpolypeptide variant having one or more amino acid additions orsubstitutions or internal deletions (wherein the resulting polypeptideis at least 6 amino acids or more in length) as compared to thetmst2-receptor polypeptide amino acid sequence set forth in SEQ ID NO: 8or 10, tmst2-receptor-receptor like polypeptide fragments may resultfrom alternative RNA splicing or from in vivo protease activity. Inpreferred embodiments, truncations comprise about 10 amino acids, orabout 20 amino acids or about 50 amino acids, or about 75 amino acids,or about 100 amino acids, or more than about 100 amino acids. Thepolypeptide fragments so produced will comprise about 25 contiguousamino acids, or about 50 amino acids, or about 75 amino acids or about100 amino acids, or about 150 amino acids, or about 200 amino acids.Such tmst2-receptor polypeptide fragments may optionally comprise anamino terminal methionine residue. It will be appreciated that suchfragments can be used, for example, to generate antibodies totmst2-receptor polypeptides.

The term “tmst2-receptor fusion polypeptide” refers to a fusion oftmst2-receptor polypeptide, fragment, variant, ortholog and/orderivative thereof, one or more amino acids (such as heterologouspeptide or polypeptide), preferably at the amino- or carboxy- terminusof the tmst2-receptor polypeptide as set forth in SEQ ID NO: 8 or 10. Anon-limiting example of such a fusion is a fusion between a tmst2polypeptide of the present invention and the Fc fragment of animmunoglobulin molecule. Fusion polypeptides according to the presentinvention may have for example improved stability in vivo or in vitro,improved solubility or improved circulatory half-life.

The term “tmst2-receptor polypeptide derivatives” refers totmst2-receptor polypeptides, variants, or fragments thereof, that havebeen chemically modified, as for example, by covalent attachment of oneor more water soluble polymers, N-linked or O-linked carbohydrates,sugars, phosphates, and/or other such molecules. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the purified or chide protein with an organic derivatizing agent thatis capable of reacting with selected side chains or terminal residues.The resulting covalent derivatives are also useful in programs directedat identifying residues important for biological activity. Thederivatives are modified in a manner that is different from naturallyoccurring tmst2-receptor polypeptide either in the type or location ofthe molecules attached to the polypeptide. Derivatives further includedeletion of one or more chemical groups naturally attached to thetmst2-receptor polypeptide.

The terms “biologically active tmst2-receptor polypeptides”,“biologically active tmst2-receptor polypeptide fragments”,“biologically active tmst2-receptor polypeptide variants”, and“biologically active tmst2-reccptor polypeptide derivatives” refer totmst2-receptor polypeptides having at least one activity characteristicof a tmst2-receptor polypeptide, such as the ability to bind andneutralize TNF-like ligand activity in biological assays. Immunogenicfragments of tmst2-receptor polypeptide(s) are those capable of inducingin a host animal antibodies directed to the tmst2 fragment.

The term “isolated polypeptide” refers to a polypeptidc of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates or other materials with which itis naturally found when isolated from the source cell, (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature, (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature, or (4) does not occurin nature. Preferably, the isolated polypeptide is substantially freefrom any other contaminating polypeptides or other contaminants that arefound in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

The term “tmst2-receptor-receptor like polypeptide ortholog” refers to apolypeptide from another species that corresponds to tmst2-receptorpolypeptide amino acid sequence as set forth in SEQ ID NO: 8 or 10. Forexample, mouse and human tmst2-receptor polypeptides are consideredortholog,s of each other.

The term “mature tmst2-receptor polypeptide” refers to a tmst2-receptorpolypeptide lacking a leader sequence and may also include othermodifications of a polypeptide such as proteolytic processing of theamino terminus (with or without a leader sequence) and/or the carboxyterminus, cleavage of a smaller polypeptide from a larger precursor,N-linked and/or O-linked glycosylation, and the like.

The term“mutein” refers to a mutant protein, polypeptide, variants,analogs or fragments of tmst2-receptor polypeptide. Muteins oftmst2-receptor polypeptide may be prepared by deletion, insertion,substitution, point mutation, truncation, addition, transposition, PCRamplification, site-directed mutagenesis or other methods known in theart.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The terms “effective amount” and “therapeutically effective amount”refer to the amount of a tmst2-receptor polypeptide necessary to supportan observable level of one or more biological activities of the decoyTNF-receptor polypeptides as set forth above, to bring about ameaningful patient benefit, i.e. treatment, healing, prevention, oramelioration of a condition. When applied to an individual activeingredient, administered alone, the tenn refers to that ingredientalone. When applied to combinations, the teni refers to combined amountsof active ingredients that result in therapeutic eftect, whenadministered in combination, serially or simultaneously. Thetmst2-receptor polypeptides that have use in practicing the presentinvention may be naturally occurring full length polypeptides, ortruncated polypeptides or variant homologs or analogs or derivatives orpeptide fragments. Illustrative analogs include those in which one ormore divergent amino acids between two species are substituted with thedivergent amino acid from another species. Divergent amino acids mayalso be substituted with any other amino acid whether it be aconservative or a non-conservative amino acid.

Relatedness of Nucleic Acid Molecules and/or Polypeptides

The term “identity”, as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween nucleic acid molecules or polypetides as the case may be, asdetermined by the match between two or more strings of nucleotide oramino acid sequences. “Identity” measures the percent of identicalmatches between the smaller of two or more sequences with gap alignmentsif any addressed by a particular mathematical model or computer program(i.e., “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity”, refers to a measure of similarity which includes bothidentical matches and conservative substitution matches. If twopolypeptide sequences have, for example, 10/20 identical amino acids,and the remainder are all non-conservative substitutions, then thepercent identity and similarity would both be 50%. If in the sameexample, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the per centsimilarity would be 75% (15/20). T herefore, in cases where there areconservative substitutions, the degree of similarity between twvopolypeptide sequences will be higher than the percent identity betweenthose two sequences.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015M sodium chloride, 0.0015M sodiumcitrate at 65-68° C. or 0.015M sodium chloride, 0.0015M sodium citrate,and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory,(Cold Spring Harbor, N.Y. 1989); and Anderson et al., Nutcleic AcidHybridisation: a practical approach, Ch. 4, IRL Press Limited (Oxford,England).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also beused,used; however, the rate of hybridization will be affected. Otheragents may be included in the hybridization and washing buffers for thepurpose of reducing non-specific and/or background hybridization.Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1%sodium pyrophosphate, 0.1% sodium dodecylsulfate (NaDodSO4 or SDS),ficoll, Denhardt's solution, sonicated salmon sperm DNA (or anothernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7.4,6.8-7.4; however, at typical ionic strengthconditions, the rate of hybridization is nearly independent of pH. SeeAnderson et al., Nucleic Acid Hybridisaition: a Practical Approach, Ch.4, IRL Press Limited (Oxford, England).

Factors affecting the stability of a DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:

Tm(° C.)=81.5+16.6(log[Na+])+0.41(%G+C)−600/N−0.72(%formamide)

where N is the length of the duplex formed, [Na+] is the molarconcentration of the sodium ion in the hybridization or washingsolution, %G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately” moderately stringent conditions “conditions”refers to conditions under which a DNA duplex with a greater degree ofbase pair mismatching than could occur under “highly stringentconditions” is able to form. Examples of typical “moderately” moderatelystringent conditions “conditions” are 0.015M sodium chloride, 0.001 5Msodium citrate at 50-65° C. or 0.015M sodium chloride, 0.0015M sodiumcitrate, and 20% formamide at 37-50° C. By way of example, a “moderatelystringent” “moderately stringent” condition of 50° C. in 0.015M sodiumion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature orraise the ionic strength.

A good estimate of the melting temperature in 1M NaCI* foroligonucleotide probes up to about 20 nt is given by:

Tm=2° C. per A−T base pair+4° C. per G−C base pair

*The sodium ion concentration in 6×salt sodium citrate (SSC) is 1M. SeeSuggs et al., Developiential Biology Using Purified Genes, p. 683, Brownand Fox (eds.) (1981). High stringency washing conditions foroligonucleotides arc usually at a temperature of 0-5° C. below the Tm ofthe oligonucleotidc in 6×SSC, 0.1% SDS.

The term “conservative amino acid substitution” refers to a substitutionof a native amino acid residue with a nonnative residue such that thereis little or no effect on the polarity or charge of the amino acidresidue at that position. For example, a conservative substitutionresults from the replacement of a non-polar residue in a polypeptidewith any other non-polar residue. Further, any native residue in thepolypeptide may also be substituted with alaninie, as has beenpreviously described for “alaninie scanning mutagenesis”. General rulesfor conservative amino acid substitutions are set forth in Table I.

TABLE I Conservative Amino Acid Substitutions Uncharged Basic: Acidic:Polar: Non-Polar: argininc glutamic acid glutamine phenylalanine valinelysine aspartic acid asparagine tryptophan proline histidine serinecysteine methionine threonine glycine leucine tyrosine alaninenorleucine soleticine

Conservative modifications to the amino acid sequence (and thecorresponding modifications to the encoding nucleotides) are expected toproduce tmst2-receptor having functional and chemical characteristicssimilar to those of naturally occurring tmst2-receptor. In contrast,substantial modifications in the functional and/or chemicalcharacteristics of tmst2-receptor may be accomplished by selectingsubstitutions that differ significantly in their effect on maintaining(a) the structure of the molecular backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain.

Naturally occurring residues may be divided into groups based on commonside chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gln, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced into regions of the human tmst2-receptormolecule that arc homologous with non-human tmst2-receptor or into thenon-homologous regions of the molecule.

Conservative amino acid substitutions also encompass non naturallyoccurring amino acid residues which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

In making such changes, the hydropathic index of amino acids may beconsidered. Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparaginc (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functionally equivalent protein orpeptide thereby created is intended for use in immunologicalembodiments, as in the present case. The greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property to the protein.

The following hydrophilicity values have been assigned to amino acidresidues: argininie (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidinie(−0.5); cysteine (−1.0); methioninie (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan.(−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.One may also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of thetmst2-receptor-receptor like polypeptide, or to increase or decrease theaffinity of the tmst2-receptor-receptor like polypeptides describedherein.

Exemplary amino acid substitutions are set forth in Table II.

TABLE II Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe LysArg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Leu Ala, Norleucine

A skilled artisan will be able to deteniine suitable variants of thepolypeptide as set forth in SEQ ID NO: 8 or 10 using well knowntechniques. For identifying suitable areas of the molecule that may bechanged without destroying activity, one skilled in the art may targetareas not believed to be important for activity. For example, whensimilar polypeptides with similar activities from the same species orfrom other species are known, one skilled in the art may compare theamino acid sequence of an tmst2-receptor polypeptide to such similarpolypeptides. With such a comparison, one can identify residues andportions of the molecules that are conserved among similar polypeptides.It will be appreciated that changes in areas of an tmst2-receptorpolypeptide that are not conserved relative to such similar polypeptideswould be less likely to adversely affect the biological activity and/orstructure of the tmst2-receptor polypeptide. One skilled in the artwould also know that, even in relatively conserved regions, one maysubstitute chemically similar amino acids for the naturally occurringresidues while retaining activity (conservative amino acid residuesubstitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in an tmst2-reccptor-receptor likepolypeptide that correspond to amino acid residues that are importantfor activity or structure in similar polypeptides. One skilled in theart may opt for chemically similar amino acid substitutions for suchpredicted important amino acid residues of tmst2-receptor polypeptides.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of that information, one skilled in the art maypredict the alignment of amino acid residues of an tmst2-receptorpolypeptide with respect to its three dimensional structure. One skilledin the art may choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays know to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change would be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where luither substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Opin. in Biotech., 7(4);422-427 (1996), Chou et al. Biochemnistry, 13(2):222-245 (1974); Chou etal. Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol.Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev.Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979).Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Opin. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

Additional methods of predicting secondary structure include“thireading” (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997);Sippl et al, Structure, 4(1):15-9 (1996)), “profile analysis” (Bowie etal., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzyn.,183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci. USA,84(13):4355-4358 (1987)), and “evolutionary linkage” (See Home, supra,and Brenner, supra).

Preferred tmst2-receptor-receptor like polypeptide variants includeglycosylation variants wherein the number and/or type of glycosylationsites has been altered compared to the amino acid sequences set forth inSEQ ID NO: 8 and 10. In one embodiment, tmst2-receptor polypeptidevariants comprise a greater or a lesser number of N-linked glycosylationsites than the amino acid sequences set forth in SEQ ID NO: 8 and 10. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr vlwhercin the amino acid residue designated as X may be anyamino acid residue except proline. The substitution(s) of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain.

Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. Also provided is arearrangemenit of N-linked carbohydrate chains wherein one or moreN-linked glycosylation sites (typically those that are naturallyoccurting) are eliminated and one or more new N-linkcd sites arecreated. Additional preferred tmst2-receptor variants include cysteinevariants, wherein one or more cysteine residues are deleted from orsubstituted for another amino acid (e.g., serine) as compared to theamino acid sequences set forth in SEQ ID NO: 8 and 10. Cysteine variantsare useful when tmst2-receptor polypeptides must be refolded into abiologically active conformation Such as after the isolation ofinsoluble inclusion bodies. Cysteine variants generally have fewercysteine residues than the native protein, and typically have an evennumber to minimize interactions resulting from unpaired cystcines.

In addition, the polypeptide comprising the amino acid sequence of SEQID NO: 8 or 10 or a tmst2-receptor polypeptide variant may be fused to ahomologous polypeptide to form a homodimer or to a heterologouspolypeptide to form a heterodimer. Heterologous peptides andpolypeptides include, but are not limited to: an epitope to allow forthe detection and/or isolation of an tmst2-receptor fusion polypeptide;a transmembrane receptor protein or a portion thereof, such as anextracellular domain, or a transmembrane and intracellular domain; aligand or a portion thereof which binds to a transmembrane receptorprotein; an enzyme or portion thereof which is catalytically active; apolypeptide or peptide which promotes oligomerization, such as a leucinezipper domain; a polypeptide or peptide which increases stability, suchas an immunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO: 8 or 10 or antmst2-receptor-receptor like polypeptide variant.

Fusions can be made either at the amino terminus or at the carboxyterminus of the polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 8 or 1 0 or an tmst2-receptor polypeptide variant. Fusionsmay be direct with no linker or adapter molecule or indirect Lisin alinker or adapter molecule. A linker or adapter molecule may be one ormore amino acid residues, typically up to about 20 to about 50 aminoacid residues. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for a protease toallow for the separation of the fused moieties. It will be appreciatedthat once constructed, the fusion polypeptides can be derivatizedaccording to the methods described herein.

In a further embodiment of the invention, the polypeptide comprising theamino acid sequence of SEQ ID NO: 8 or 10 or a tmst2-receptorpolypeptide variant is fused to one or more domains of an Fc region ofhuman IgG. Antibodies comprise two functionally independent parts, avariable domain known as “Fab”, which binds antigen, and a constantdomain known as “Fc”, which is involved in effector functions such ascomplement activation and attack by phagocytic cells. An Fc has a longserum half-life, whereas an Fab is short-lived. Capon et al., Nature,337:525-31 (1989). When constructed together with a therapeutic protein,an Fc domain can provide longer half-life or incorporate such functionsas Fc receptor binding, protein A binding, complement fixation andperhaps even placental transfer. Id. Table III summarizes the use ofcertain Fc fusions known in the art.

TABLE III Fc Fusion with Therapeutic Potential Form of FusionTherapeutic Fc partner implications Reference IgG1 N-terminus Hodgkin'sU.S. Pat. No. of CD30-L disease; 5,480,981 anaplastic lyphoma; T- cellleukemia Murine IL-10 anti-inflamma- Zheng et al. (1995), J. Fcg2a tory;transplant Immunol. 154: 5590- rejection 5600 IgG1 TNF septic shockFisher et al. (1996), N. receptor Engl. J. Med., 334: 1697- 1702; VanZee et al., (1996), J. Immunol., 156: 2221-2230 IgG, IgA, TNFinflammation, U.S. Pat. No. 5,808,029, IgM, or receptor autoimmuneissued Sept. 15, IgE disorders 1998 (excluding the first domain) IgG1CD4 AIDS Capon et al. (1989), receptor Nature 337: 525-531 IgG1,N-terminus anti-cancer, Harvill et al. (1995), IgG3 of IL-2 antiviralImmunotech., 1: 95-105 IgG1 C-terminus osteoarthritis; WO 97/23614,published of OPG bone density Jul. 3, 1997 IgG1 N-terminus anti-obesityPCT/US 97/23183, filed of leptin Dec. 11, 1997 Human Ig CTLA-4autoimmune Linsley (1991), J. Exp. Cg1 disorders Med., 174: 561-569

In one example, all or a portion of the human IgG hinge, CH2 and CH3regions may be fused at either the N-terminus or C-terminius of thetmst2-receptor-receptor like polypeptides using methods known to theskilled artisan. The resulting tmst2-receptor fusion polypeptide may bepurified by use of a Protein A affinity column. Peptides and proteinsfused to an Fc region have been found to exhibit a substantially greaterhalf-life in vivo than the unfused counterpart. Also, a fusion to an Fcregion allows for dimerization/mutltimerization of the fusionpolypeptide. The Fc region may be a naturally occurring Fc region, ormay be altered to improve certain qualities, such as therapeuticqualities, circulation time, reduce aggregation, etc.

Identity and similarity of related nucleic acid molecules andpolypeptides can be readily calculated by known methods. Such methodsinclude, but are not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, (New York,1988); Biocomputing: Informatics and Genosme Projects, Smith, D. W.,ed., Academic Press, (New York, 1993); Computer Analysis of SequenceData, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humania Press,(New Jersey, 1994); Sequence Analysis in Molecular Biology, von Heinje,G., Academic Press, (1987); Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M. Stockton Press, (New York, 1991); and Carillo etal., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity and/or similarity are designedto give the largest match between the sequences tested. Methods todetermine identity and similarity are described in publicly availablecomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, the GCG program package, including GAP (Devereux, et al.,Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Atschul, et al., J. Molec. Biol. 215:403-410 (1990). The BLAST Xprogram is publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul, et al. NCBNLM NIH Bethesda, Md. 20894; Altschul, et al., J. Mol. Biol. 215:403-410(1990). The well known Smith Waterman algorithm may also be used todetermine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in the matching of only a short region of the two sequences, andthis small aligned region may have very high sequence identity eventhough there is no significant relationship between the two full lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method (GAP program) will result in an alignment that spans atleast 50 contiguous amino acids of the target polypeptide.

For example, Usitlg the computer algorithm GAP (Genetics Computer Group,(University of Wisconsin, Madison, Wis.), two polypeptides for which thepercent sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal; the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually {fraction (1/10)} times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. A standard comparison matrix (see Dayhoff et al, in:Atlas of Proteint Sequtence and Structure, vol. 5, supp.3 (1978) for thePAM 250 comparison matrix; see Henikoff et al., Proc. Natl. Acad. SciUSA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is alsoused by the algorithm.

Preferred parameters for a polypeptide sequence comparison include thefollowing:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970),Comparison matrix: BLOSUM 62 from Henikoff and Hienikoff, Proc. Natl.Acad. Sci. USA 89 10915-10919 (1992).

Gap Penalty: 12

Gap Length Penalty: 4

Threshold of Similarity: 0

The GAP program is useful with the above parameters. The aforementionedparameters are the default parameters for polypeptide comparisons (alongwith no penalty for end gaps) using the GAP algorithm.

Preferred parameters for nucleic acid molecule sequence comparisonsinclude the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970)

Comparison matrix: matches+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

The GAP program is also useful with the above parameters. Theaforementioned parameters are the default parameters for nucleic acidmolecule comparisons.

Other exemplary algorithms, gap opening penalties, gap extensionpenalties, comparison matrices, thresholds of similarity, etc. may beused by those of skill in the art, including those set forth in theProgram Manual, Wisconsin Package, Version Sep. 9, 1997. The particularchoices to be made will depend on the specific comparison to be made,such as DNA-to-DNA, protein-to-protein, protein-to-DNA; andadditionally, whether the comparison is between given pairs of sequences(in which case GAP or BestFit are generally preferred) or between onesequence and a large database of sequences (in which case FASTA orBLASTA are preferred).

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two ftill lengthsequences. Accordingly, in a preferred embodiment, the selectedalignment method will result in an alignment that spans at least about66 contiguous amino acids of the claimed full length polypeptide.

Synthesis

It will be appreciated by those skilled in the art the nucleic acid andpolypeptide molecules described herein may be produced by recombinantand other means.

Nucleic Acid Molecules

The nucleic acid molecules encode a polypeptide comprising the aminoacid sequence of an tmst2 like polypeptide can readily be obtained in avariety of ways including without limitation, chemical synthesis, cDNAor genomic library screening, expression library screening and/or PCRamplification of cDNA.

Recombinant DNA methods used herein are gYenerally, but not limited to,those set forth in Sambrook et al. (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)) and/or Ausubel et al., eds., (Current Protocols in MolecularBiology, Green Publishers Inc. and Wiley and Sons, NY (1994)).

The present invention provides for nucleic acid molecules as describedherein and methods for obtaining the molecules. A gene or cDNA encodinga “tmst2 receptor polypeptide” or fragment thereof may be obtained byhybridization screening of a genomic or cDNA library, or by PCRamplification. Probes or primers useful for screening a library byhybridization can be generated based on sequence infornation for otherknown genes or gene fragments fromt the same or a related family ofgenes, such as, for example, conserved motifs. In addition, where a geneencoding tmst2-receptor polypeptide has been identified from onespecies, all or a portion of that gene may be used as a probe toidentify corresponding genes from other species (orthologs) or relatedgenes from the same species (homologs). The probes or primers may beused to screen cDNA libraries from various tissue sources believed toexpress the “tmst2-receptor gene”.

In addition, part or all of a nucleic acid molecule having the sequenceas set forth in SEQ ID NO: 7 or 9 may be used to screen a genomiclibrary to identify and isolate a gene encoding “tmst2 receptor.”Typically, conditions of moderate or high stringency will be employedfor screening to minimize the number of false positives obtained fromthe screen.

The availability of the cDNA coding for the tmst2-receptor or fractionsthereof is the prerequisite for obtaining the genomic DNA. Understringent conditions, a DNA library is screened and the clones obtainedare investigated to see whether they contain the regulatory sequenceelements needed for gene expression in addition to the coding regions(e.g. checking for promoter function by fusion with coding regions ofsuitable reporter genes). Methods for screening DNA libraries understringent conditions are taught, for example, in EPA 0 174 143,incorporated herein by reference. Obtaining, the genomic DNA sequencemakes it possible to investigate the regulatory sequences situated inthe area which does not code for the “tmst2-receptor”, particularly inthe 5′-flanking region, for any possible interaction with knownsubstances which modulate gene expression, e.g. transcription factors orsteroids, or possibly discover new substances which might have aspecific effect on the expression of this gene. The results of suchinvestigations provide the basis for the targeted use of such substancesfor modulating tmst2-receptor expression and hence for directlyinfluenicing, the ability of the cells to interact with TNF family ofligands. As a result the specific reaction with the ligands and theresulting effects can be suppressed.

The scope of the present invention also includes DNAs which code forsubtypes of the tmst2-receptor or its soluble forms, which may haveproperties different from those of the present tmst2-receptor. These areexpression products which are formed by alternative splicing and havemodified structures in certain areas, e.g. structures which can bringabout a change in the affinity and specificity for the ligand or achange in terms of the nature and efficiency of signal transmission.

With the aid of the cDNA coding for the tmst2-receptor it is possible toobtain nucleic acids which hybridize with the cDNA or fragments thereofunder conditions of low stringency and code for a polypeptide capable ofbinding TNF-related ligands or contain the sequence coding for such apolypeptide.

According to a further aspect, the invention relates to recombinanttmst2-receptor polypeptide(s), preferably in a secretable form, whichconstitutes the soluble part of the tmst2-receptor. The invention alsocontemplates the production of a soluble fonn of the tmst2-receptor,which is secreted into the cell supernatant, by recombinant DNAtechnology wherein the DNA coding for tmst2-receptor with a sequencecoding for a signal peptide under the control of a suitable promoter isintroduced into suitable host organisms, especially eukaryotic andpreferably higher eukaryotic cells.

Nucleic acid molecules encoding tmst2-receptor polypeptides may also beidentified by expression cloning which employs the detection ofpositiveclones based upon a property of the expressed protein. Typically,nucleic acid libraries are screened by binding of an antibody or otherbinding partner (e.g., receptor or ligand) to cloned proteins which areexpressed and displayed on a host cell surface. The antibody or thebinding partner is modified with a detectable label to identify thosecells expressing the desired clone.

Two murine TNF receptors, tmst2-receptor described herein, andymkz5-receptor (cloned by Amgen), have been identified to be closelylinked within the murine genome. Both of these novel receptors bindTRAIL in a species specific manner (See Example 9). Therefore. thecharacterization of the murine genes, tmst2 and ymkz5, may aid in thediscovery of human TRAIL decoy receptors based on functionality and notsolely based on primary sequence homology. Identification of humantmst2/ymkz5 orthologs of the invention will be facilitated bychromosomal and structural studies to reveal two highly related geneswhich are closely linked on the chromosome, one which is GPI-linked andthe other a transmembrane receptor. Alternatively, the human genome mayonly harbor one ortholog which may be identified in the region syntenicwith the mouse tmst2/ymkz5 locus.

Recombinant expression techniques conducted in accordance with thedescriptions set forth below may be followed to produce thesepolynucleotides and to express the encoded polypeptides. For example, byinserting a nucleic acid sequence which encodes the amino acid sequenceof a tmst2-receptor polypeptide into an appropriate vector, one skilledin the art can readily produce large quantities of the desirednucleotide sequence. The sequences can then be used to generatedetection probes or amplification primers. Alternatively, apolynucleotide encoding the amino acid sequence of a tmst2-receptorpolypeptide can be inserted into an expression vector. By introducingthe expression vector into an appropriate host, the encodedtmst2-receptor polypeptide may be produced in large amounts.

Another method for obtaining a suitable nucleic acid sequence is thepolyierase chain reaction (PCR). In this method, cDNA is prepared frompoly(A)+RNA or total RNA using the enzyme reverse transcriptase. Twoprimers, typically complementary to two separate regions of cDNA(oligonucleotides) encoding the amino acid sequence of an tmst2-receptorpolypeptide, are then added to the cDNA along with a polymerase such asTaq polymerase, and the polymerase amplifies the cDNA region between thetwo primers.

Another means of preparing a nucleic acid molecule encoding a varianttmst2-receptor polypeptide or a biologically active fragment thereof isby chemical synthesis using methods well known to the skilled artisansuch as those described by Engels et al.(Angew. Chem. Intl. Ed.,28:716-734 (1989)). These methods include, inter alia, thephosphotriester, phosphoramidite, and H-phosphoniate methods for nucleicacid synthesis. A preferred method for such chemical synthesis ispolymer-supported synthesis using standard phosphoramiditc chemistry.Typically. the DNA encoding the tmst2-receptor polypeptide will beseveral hundred nucleotides in lengtth. Nucleic acids larger than about100 nucleotides can be synthesized as several fragments using thesemethods. The fragments can then be ligated together to form the fulllength tmst2-receptor polypeptide. Usually, the DNA fragment encodingthe amino terminus of the polypeptide will have an ATG, which encodes amethionine residue. This methionilne may or may not be present on themature form of the tmst2-receptor polypeptide, depending on whether thepolypeptide produced in the host cell is designed to be secreted fromthat cell.

In some cases, it may be desirable to prepare nucleic acid moleculesencoding tmst2-receptor polypeptide variants or muteins. Nucleic acidmolecules encoding variants may be produced using site directedmutagenesis, transposition, deletion, addition, truncation, PCRamplification, or other appropriate methods, where the primer(s) havethe desired point mutations (see Sambrook et al., supra, and Ausubel etal., supra, for descriptions of mutagenesis techniques), provided thatDNA's modified in this way code for polypeptides capable of binding oneor more members of the TNF-family. Chemical synthesis using methodsdescribed by Engels et al., supra, may also be used to prepare suchvariants. Other methods known to the skilled artisan may be used aswell.

In certain embodiments, nucleic acid variants contain codons which havebeen altered for the optimal expression of a tmst2-receptor polypeptidein a given host cell. Particular codon alterations will depend upon thetmst2-receptor polypeptide(s) and host cell(s) selected for expression.Such “codon optimization” can be carried out by a variety of methods,for example, by selecting codons which are preferred for use in highlyexpressed genes in a given host cell. Computer algorithms whichincorporate codon frequency tables such as “Ecohigh. cod” for codonpreference of highly expressed bacterial genes may be used and areprovided by the University of Wisconsin Package Version 9.0, GeneticsComputer Group, Madison, Wis. Other useful codon frequency tablesinclude “Celecans_high.cod”, “Celegans_low.cod”, “Drosophila_high.cod”,“Human_high.cod”, “Maize_high.cod”, and “Yeast_high.cod”.

In other embodiments, nucleic acid molecules encode tmst2-receptorvariants with conservative amino acid substitutions as defined above,tmst2 receptor variants comprising an addition and/or a deletion of oneor more N-linked or O-linked glycosylationi sites, or tmst2-receptorpolypeptide fragments as described above. In addition, nucleic acidmolecules may encode any combination of tmst2-receptor variants.fragments, and fusion polypeptides described herein provided that DNA'smodified in this way code for polypeptides capable of finding one ormore members of TNF super gene family of ligands and receptors.

Expression of tmst2 in Eukaryotic and Prokaryotic Cells

A nucleic acid molecule encoding a tmst2-receptor polypeptide may beinserted into an appropriate expression vector using standard ligationtechniques. The vector is typically selected to be functional in theparticular host cell employed (i.e., the vector is compatible with thehost cell machinery such that amplification of the gene and/orexpression of the gene can occur). A nucleic acid molecule encoding atmst2-reccptor polypeptide may be amplified/expressed in prokaryotic,yeast, insect (baculovirus systems) and/or eukaryotic host cells.Selection of the host cell will depend in part on whether thetmst2-receptor polypeptide is to be post-translationally modified (e.g,glycosylated and/or phosphorylated). lfso, yeast, insect, or mammalianhost cells are preferable. For a reveiw of expression vector, see Meth.Enz. vol. 185, D.V. Goeddel ed. Academic Press, Inc., San Diego, Calif.(1990).

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone of the following nucleotides: a promoter, one or more enhancersequences, an origin of replication, a transcriptional terminationsequence, a complete intron sequence containing a donor and acceptorsplice site, a leader sequence for secretion, a ribosome binding site, apolyadenylation sequence, a polylinker region for inserting the nucleicacid encoding the polypeptide to be expressed, and a selectable markerelement. Each of these sequences is discussed below.

Optionally, the vector may contain a “tag” sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of thetmst2-receptor polypeptide coding sequence; the oligonucleotide moleculeencodes polyHis (such as hexaHis), or other “tag” such as FLAG, HA(hemaglutinin Influenza virus) or myc for which commercially availableantibodies exist. Optionally, the tmst2 gene can also be fused in frameat the N-terminal for example to an IgG Fc region. This tag is typicallyfused to the polypeptide upon expression of the polypeptide, and canserve as means for affinity purification of the tmst2-receptorpolypeptide from the host cell althougLh it may also prolong thecirculatory half life of a tmst2 polypeptide. Affinity purification canbe accomplished, for example, by column chromatography using antibodiesor protein-A column against the tag as an affinity matrix. Optionally,the tag can subsequently be removed from the purified tmst2-receptorpolypeptide by various means such as using certain peptidases forcleavage.

The 5′-flanking region of a gene contains a nucleic acid sequence towhich RNA polymerase binds and initiates transcription. This nucleicsequences, known as the promoter region, determines both the nature ofthe enzyme that attaches to it and the rate of RNA synthesis. A numberof eukarvotic and prokaryotic promoter elements are known in the art andare used to enhance gene transcription. Flanking sequneces may behomologous (i.e., from the same species and/or strain as the host cell),heterologous (i.e, from a species other than the host cell species orstrain), hybrid (i.e., a combination of gene flanking sequences frommore than one source), or synthetic, or the flanking sequences may benative sequences which normally function to regulate tmst2-receptorexpression. As such, the source of a flanking sequence may be anyprokaryotie or eukaryotic organism, any vertebrate or invertebrateorganism, or any plant, provided that the flanking sequences isfunctional in, and can be activated by, the host cell machinery.

The flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein other than the endogenoLstmst2-receptor gene flanking sequence will have been previouslyidentified by mapping and/or by restriction endonuclease digestion andcan thus be isolated from the proper tissue source using the appropriaterestriction endonucleases. In some cases, the full nucleotide sequenceof one or more flanking sequence may be known. Here, the flankingsequence may be synthesized using the methods described above fornucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with suitableoligonucleotide and/or flanking sequence fragments from the same oranother species.

Where the flanking sequence is not known, a fragmnent of DNA containinga flanking sequence may be isolated from a larger piece of DNA that maycontain, for example, a coding sequence or even another gene or genes.Isolation may be accomplished by restriction endonuclease digestion toproduce the proper DNA fragment followed by isolation using agarosc gelpurification, QIAGEN® column chromatography (Chatsworth, Calif.). orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origen aids in theamplification of the vector in a host cell. Amplification of the vectorto a certain copy number can, in some cases, be important for theoptimal expression of the tmst2-receptor polypeptide. If the vector ofchoice does not contain an origin of replication site, one may bechemically synthesized based on a known sequence, and ligated into thevector. For example, the origin of replication from the plasmid pBR322(Product No. 303-3s, New England Biolabs, Beverly, Mass.) is suitablefor most Gram-negative bacteria and various origins (e.g., SV 40,polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammaliancells. Generally, the origin of replication component is not needed formammalian expression vectors (for example, the SV40 origin is often usedonly because it contains the early promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such asthose described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may be used forselection in prokaryotic and eukaryotic host cells.

Other selection g,enes may be used to amplify the gcne which will beexpressed. Amplification is the process wherein genes which are ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable, amplifiable, selectable markersfor mammalian cells include dihydrofolate reductase (DHFR) and thymidilekinase. The mammalian cell transformants are placed under selectionpressure which only the tranisformants are uniquely adapted to surviveby virtue of the marker present in the vector. Selection pressure isimposed by culturing the transformed cells under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to the amplification of both thes election gene and theDNA that encodes tmst2-receptor. As a result, increased quantities oftmst2-receptor polypeptide are synthesized from the amplified DNA.

A ribosome binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the tmst2-receptorpolypeptide to be expressed. The Shine-Dalgarno sequence is varied butis typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

A leader, or signal, sequence may be used to direct the secretion oftmst2-receptor polypeptide out of the host cell where it is synthesized.Typically, the signal sequence is positioned in the coding region of thetmst2-receptor nucleic acid molecule, or directly at the 5′ end of thetmst2-receptor polypeptide coding region. Many signal sequences havebeen identified, and any ofthose that are functional in the selectedhost cell may be used in conjunction with the tmst2-receptor gene orcDNA. Therefore, a signal sequence may be homologous (naturallyoccurring) or heterologotis to the tmst2-receptor gene or cDNA, and maybe homologous or heterologous to the tmst2-receptor gene or cDNA.Additionally, a signal sequence may be chemically synthesized usingmethods described herein. In most cases, the secretion of antmst2-receptor polypeptide from the host cell via the presence of asignal peptide will result in the removal of the signal peptide from thetmst2-receptor polypeptide.

The signal sequnce may be a component of the vector, or it may be a partof tmst2-reccptor DNA that is inserted into the vector. The nativetmst2-receptor DNA encodes a signal sequence at the amino terminus ofthe protein that is cleaved during post-translational processing of themolecule to form the mature tmst2-receptor protein product. Includedwithin the scope of this invention are tmst2-reccptor nucleotides withthe native signal sequence as well as tmst2-receptor nucleotides whereinthe native signal sequence is replaced with, a heterologous signalsequence joined to tmst2-receptor polypeptide coding region. Theheterologous signal sequence selected should be one that is recognizedand processed, i.e., cleaved by a signal peptidase, by the host cell.For prokaryotic host cells that do not recognize and process the nativetmst2-receptor signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, or heat-stable enterotoxin IIleaders. For yeast secretion, the native tmst2-receptor signal sequencemay be substituted by the yeast invertase, alpha factor, or acidphosphatase leaders. In mammalian cell expression the native signalsequence of the tmst2-receptor polypeptide is satisfactory, althoughother mammalian signal sequences may be suitable.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a particular signal peptide, or addpresequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acid residuesincident to expression, which may not have been totally removed. Forexample, the final protein product may have one or two amino acids foundin the peptidase cleavage site, attached to the N-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired tmst2-receptor polypeptide, ifthe enzyme cuts at such area within the mature polypeptide.

In many cases, transcription of a nucleic acid molecule is increased bythe presence of one or more introns in the vector; this is particularlytrue where a polypeptide is produced in eukaryotic host cells,especially mammalian host cells. The introns may be naturally occurringwithin the tmst2-reccptor uene, especially where the gene used is a fulllength genomic sequence or a frament thereof. Where the intron is notnaturally occurring within the gene (as for most cDNAs), the intron(s)may be obtained from another source. The position of the intron withrespect to 5′-flanking sequences and the tmst2-receptor gene isgenerally important, as the intron must be transcribed to be effective.Thus, when an tmst2-receptor cDNA molecule is being, expressed, thepreferred position for the intron is 3′ to the transcription start site,and 5′ to the polyA transcription termination sequence. Preferably, theintron or introns will be located on one side or the other (i.e., 5′ or3′) of the cDNA such that it does not interrupt the this codingsequence. Any intron from any source, including any viral, prokaryoticand eukaryotic (plant or animal) organisms, may be used to practice thisinvention, provided that it is compatible with the host cell(s) intowhich it is inserted. Also included herein are synthetic introns.Optionally, more than one intron may be used in the vector.

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to the molecule encoding the tmst2-receptor protein.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of a particular molecule, suchas that encoding tmst2-receptor. Promoters are conventionally groupedinto one of two classes, inducible promoters and constitutive promoters.Inducible promoters initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,such as the presence or absence of a nutrient or a change intemperature. Constitutive promoters, on the other hand, initiatecontinual gene product production; that is, there is little or nocontrol over gene expression. A large number of promoters, recognized bya variety of potential host cells, are well known. A suitable promoteris operably linked to the DNA encoding tmst2-receptor by removing thepromoter from the source DNA by restriction enzyme digestion andinserting the desired promoter sequence into the vector. The nativetmst2-reccptor promoter sequence may be used to direct amplificationand/or expression of tmst2-receptor encoding DNA. A heterologouspromoter is preferred, however, if it permits greater transcription andhigher yields of the expressed protein as compared to the nativepromoter, and if it is compatible with the host cell system that hasbeen selected for use.

Promoters suitable for use with prokaryotic hosts include, but are notlimited to the beta-lactamase and lactose promoter systems; alkalinephosphatase, a tryptophan (trp) promoter system; and hybrid promoterssuch as the tac promoter. Other known bacterial promoters are alsosuitable. Their sequences have been published, thereby enabling oneskilled in the art to ligate them to the desired DNA sequencc(s), usinglinkers or adapters as needed to supply any useful restriction sites.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, herpes virus and mostpreferably Simian Virus 40 (SV40). Other suitable mammalian promotersinclude heterologous mammalian promoters, e.g., heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest in controlling tmst2expression include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature, 290:304-310 (1981)); the CMVpromoter; the promoter contained in the 3′ long terminal repeat (LTR) ofRous sarcoma virus (RSV) (Yamamoto et al., Cell, 22:787-797 (1980)); theherpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.U.S.A., 78:144-1445 (1981)); the regulatory sequences of themetallothionine gene (Brinster et al., Nature, 296:39-42 (1982));prokaryotic expression vectors such as the beta -lactamase promoter(Villa-Kamaroff et al., Proc. Natl. Acad. Sci. U.S.A., 75:3727-3731(1978)); or the tac promoter (DeBoer et al., Proc. Nati. Acad. Sci.U.S.A., 80:21-25 (1983)). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgcnic animals: the elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al., Cell,38:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409, (1986); MacDonald, Hepatology, 7:425-515 (1987)); theinsulin gene control region which is active in pancreatic beta cells(Hanahan, Nature, 315:115-122 (1985)); the immunoglobulin gene controlregion which is active in lymphoid cells (Grosschedl et al., Cell,38:647-658 (1984); Adames et a., Nature, 318:533-538 (1985); Alexanderet al., Mol. Cell. Biol., 7:1436-1444 (1987)); the mouse mammary tumorvirus control region which is active in testicular, breast, lymphoid andmast cells (Leder et al, Cell, 45:485-495 (1986)), the albumin genecontrol region which is active in liver (Pinkert et al., Genes andDevel., 1:268-276 (1987)); the alpha-feto-protein gene control regionwhich is active in liver (Krumlauf et al., Mol. Cell. Biol., 5:1639-1648(1985); Hammer et al., Science, 235:53-58 (1987)); the alpha1-antitrypsin gene control region which is active in the liver (Kelseyet atl., Genes and Devel., 1:161-171(1987)); the beta-globin genecontrol region which is active in myeloid cells (Mogram et al., Nature,315:338-340 (1985); Kollias et al., Cell, 46:89-94 (1986)); the myelinbasic protein gene control region which is active in oligodenidrocytecells in the brain (Readhead et al., Cell, 48:703-712, (1987)); themyosin light chain-2 gene control region which is active in skeletalmuscle (Sani, Nature, 314:283-286 (1985)); and the gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason et al., Science, 234:1372-1378 (1986)).

An enhancer sequence may be inserted into the vector to increase thetranscription of a DNA encoding a tmst2-receptor polyepeptode by highereukaryotes. Enhancers are cis-acting elements of DNA, usually about10-300 bp in length, that act on the promoter to increase itstranscription. Enhancers are relatively orientation and positionindependent. They have been found 5′ and 3′ to the transcription unit.Several enhancer sequences available from mammalian genes are known(e.g., globin, clastase, albumin, alpha-feto-protein and insulin).Typically, however, an enhancer from a virus will be used. The SV40enhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer, and adenovirus enhancers are exemplary enhancing elements forthe activation or upregulation of eukaryotic promoters. While anenhancer may be spliced into the vector at a position 5′ or 3′ totmst2-receptor DNA, it is typically located at a site 5′ from thepromoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe desired flanking sequences are not already present in the vector,they may be individually obtained and ligated into the vector. Methodsused for obtaining each of the flanking sequences are well known to oneskilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors iniclude, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, Carlsbad, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Phannacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. WO 90/14364)and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).

Additional suitable vectors include, but are not limited to cosmids,plasmids, or modified viruses, but it will be appreciated that thevector system must be compatible with the selected host cell. Suchvectors include, but are not limited to plasmids such as Bluescript®plasmid derivatives (a high copy number ColE1-based phagemid, StratageneCloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designedfor cloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit,PCR2.1® plasmid derivatives, Invitrogeni, Carlsbad, Calif.), andmammalian , yeast or virus vectors such as a baculovirus expressionsystem (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.). Therecombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, or other knowntechniques.

After the vector has been constructed and a nucleic acid moleculeencoding an tmst2-receptor polypeptide has been inserted into the propersite of the vector, the completed vector may be inserted into a suitablehost cell for amplification and/or polypeptide expression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, synthesizesan tmst2-receptor polypeptide which can subsequently be collected fromthe culture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity, such as glycosylation orphosphorylation, and case of folding into a biologically activemolecule.

Yeast and mammalian cells are preferred hosts of the present invention.The use of such hosts provides substantial advantages in that they canalso carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in these hosts.

Yeast recognize leader sequences on cloned mammalian gene products andsecrete peptides bearing leader sequences (i.e., pre-peptides).Mammalian cells provide post-translational modifications to proteinmolecules including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblastorigin such as VERO or CtO-K1, and their derivatives. For a mammalianhost, several possible vector systems are available for the expressionof the desired tmst2-receptor protein. A wide variety of transcriptionaland translational regulatory sequences may be employed, depending uponthe nature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Usefulsignals are regulatory signals which are temperature-senisitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

As widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired receptor molecule does notcontain any intervening codons which are capable of encoding amethionine (i.e., AUG). The presence of such codons results either inthe formation to a fusion protein (if the AUG codon is in the samereading frame as the desired receptor molecule encoding DNA sequence) ora frame-shift mutation (if the AUG codon is not in the same readingframe as the desired tmst2-receptor protein encoding sequence).

The expression of the tmst2-receptor proteins can also be accomplishedin procaiyotic cells. Preferred prokaryotic hosts include bacteria suchas E. coli, Bacillus, Streptomyces, Pseudoinonas, Salmonella, SetTatiaetc. The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F⁻, lambda⁻, prototrophic (ATCC27325)), and other enterobacteria (such as Salmonella typhimurium orSerratia mcarcescens), and various Pseudomonas species. The prokaryotichost must be compatible with the replicon and control sequences in theexpression plasmid.

To express the desired tmst2-receptor protein in a prokaryotic cell(such as, for example, E. coli, B. stibtilis, Pseudomonias.Streptomyces, etc.), it is necessary to operably link the desiredreceptor molecule encoding sequence to a functional prokaryoticpromoter. Such promoters may be either constitutive or, more preferably,regulatable (i.e., inducible or derepressible). Examples of constitutivepromoters include the int promoter of bacteriophage λ, and the blapromoter of the β-lactamase gene of pBR322, etc. Examples of inducibleprokaryotic promoters include the major right and left promoters, ofbacteriophage λ (P_(L) and P_(R)), the trp, recA, lacZ, lacI, gal, andtac promoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol.162:176-182 (1985)), the α-28-specific promoters of B. subtilis (Gilmanet al., Gene 32:11-20 (1984)), the promoters of the bacteriophages ofBacillus (Gryczan, In: The Molecular Biology of the Bacilli, AcademicPress, Inc., New York (1982)), and Streptomyces promoters (Ward et al.,Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewedby Glick, B. R., (J. Ind. Microbiol. 1:277-282 (1987); Cenatiempo, Y.Biochimie 68:505-516 (1986)); and Gottesman, S. Ann. Rev. Genet.18:415-442 (1984).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream from the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold, L., et al.(Ann. Rev. Microbiol. 35:365-404 (1981)).

The desired tmst2-receptor polypeptide encoding sequence and an operablylinked promoter may be introduced into a recipient prokaryotic oreukaryotic cell either as a non-replicating DNA (or RNA) molecule, whichmay either be linear or, morc preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomousreplicaitioni, the expression of the desired receptor molecule may occurthrough the transient expression of the introduced sequence.Alternatively, permanent expression may occur through the integration ofthe introduced sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. TIhe markermay complement an auxotrophy in the host (such as leu21, or ura3, whichare common yeast auxotrophic markers), biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene can either be directly linked to the DNA genesequences to be expressed, or introduced into the same cell byco-transfection.

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include, for e.g. the case with which recipient cells thatcontain the vector may be recognized and selected from those recipientcells which do not contain the vector; the number of copies of thevector which are desired in a particular host; and whether it isdesirable to be able to “shuttle” the vector between host cells ofdifferent species.

Any of a series of yeast gene expression systems can also be utilized.Examples of such expression vectors include the yeast 2-micron circle,the expression plasmids YEP13, YVP and YRP, etc., or their derivatives.Such plasmids are well known in the art (Botstein et al., Miami Wntr.Symp. 19:265-274 (1982); Broach, J. R., In: The Molecular Biology of theYeast Saccharotiyces: Lift Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p.445-470 (1981); Broach, Cell28:203-204 (1982)).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper or the like. The selectablemarker gcne can either be directly linked to the DNA sequences to beexpressed, or introduced into the same cell by co-transformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,Mol. Cell. Biol. 3:280 (1983), and others. Preferred eukaryotic vectorsinclude PWLNEO, PSV2CAT, POG44, PXT1, pSG, pSVK3, pBPV, pMSG, pSVL(Pharmacia).

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli such as, for example, pBR322, ColE1, pSC101,pACYC 184, πVX, pQE70, pQE60, pQE9, pBG, pD10, Phage script, psix174,pbmescript SK, pbsks, pNH8A, pNHIBa, pNH18A, pNH46A (SL rare gone),ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Such plasmids are, forexample, disclosed by Maniatis, r., et al. (In: Molecular Cloning, ALaboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor, (N.Y.1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Suchplasmids are disclosed by Gryczan, T. (In: The Molecular Biology of theBacilli, Academic Press, (New York 1982), pp. 307-329). SuitableStreptomyces plasmids include pISJ101 (Kendall et al., J Bacteriol169:4177-4183 (1987)), and Streptomyces bacteriophages such as φC31(Chater et al., In: Sixyth International Symposium on ActinotmycetalesBiology. Akademiai Kaido, Budapest, Hungary (1986), pp 45-541).Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis.8:693-704 (1986), and Izaki (Jpn. J. Bacteriol. 33:729-742 (1978)).However, any other plasmid or vector may be used as long as they arereplicable and viable in the host cell.

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced into anappropriate host. Various techniques may be employed, such as aprotoplast fusion, calcium phosphate precipitation, electroporation orother conventional techniques. After the fusion, the cells are grown inmedia and screened for appropriate activities. Expression of thesequence results in the production othfie tmst2 receptor protein.

Suitable host cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO; ATCC No. CCL-61), human embryonickidney (HEK), 293 or 293T cells (ATCC No. CRL-1573), 3T3 cells (ATCC No.CCL-92) mouse neuroblastoma N2A cells (ATCC No. CCL 131), HeLa (ATCC No.CCL-2), mouse L-929 cells (ATCC No. CCL-1), BHK (ATCC No. CCL-10) or HaK(ATCC No. CCL-15) hamster cell lines. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines, are the monkeyCOS-1 (ATCC No. 1650) and COS-7 (ATCC No. CRL-1651) cell lines, and theCV-1 cell line (ATCC No. CCL-70). Further exemplary mammalian host cellsinclude primate cell lines and rodent cell lines, including transformedcell lines. Normal diploid cells, cell strains derived from in vitroculture of primary tissue, as well as primary explants, are alsosuitable. Candidate cells may be genotypically deficient in theselection gene, or may contain a dominantly acting selection gene.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101 (ATCC No. 33694), DH5α, DH10, and MC1061(ATCC No. 53338)) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomycesspp., and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention (e.g. Sacclharomyces, Pichia, Candida, Hansenula, andTorulopsis). (Bitter, G. , Heterologous Gene Expression in Yeast in.Berger, S. L. and Kimmel, A. R., 152:673-684, (1987)). Preferred yeaststrains include, for example, Saccharomyces cerevisiae, which can betransformed readily with DNA either by preparation of spheroplasts or bytreatment with alkaline salts such as LiCl. (Itoh, I. et al. J.Bacteriol. 153:163 (1983)). Some proteins expressed in yeast cells areefficiently secreted into the culture medium while others accumulateintracellularly.

Additionally, where desired, insect cell systems may be utilized in themethods of the present invention. Such systems are described for examplein Kitts et al. (Biotechniques, 14:810-817 (1993)), Lucklow (Curr. Opin.Biotechiol., 4:564-572 (1993)) and Lucklow et al. (J. Virol.,67:4566-4579 (1993)). Preferred insect cells are Sf-9 and Hi5 cells(Invitrogen, Carlsbad, Calif.). Baculovirus vectors based on theAutographa california nuclear polyhedrosis virus, which are useful forthe introduction of genetic information into insect cells include, butare not limited to pVL1392 or 1393 (Invitrogen).

Transformation or transfection of an expression vector for atmst2-receptor polypeptide into a selected host cell may be accomplishedby methods such as calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook et al., supra.

One may also use transgenic animals to express glycosylated tmst2 likepolypeptides. For example, one may use a transgenic milk-producinganimal (a cow or goat, lor example) and obtain the present glycosylatedpolypeptide in the animal milk. One may also use plants to producetmst2-receptor polypeptides, however, in general, the glycosylationoccurring in plants is different from that produced in mammalian cells,and may result in a glycosylated product which is not suitable for humantherapeutic use.

Polypeptide Production

Host cells comprising an tmst2-reccptor expression vector (i.e.,transformed or transfected) may be cultured using standard media wellknown to the skilled artisan.

The media will usually contain all nutrients to allow for the growth andsurvival of the cells. Suitable media for culturing E. Coli cells arefor example, Luria Broth (LB) and/or Terrific Broth (TUB). SLutablemedia for culturing eukaryotic cells are Rosewell Park MemorialInstitute 1640 (RPMI 1640), Minimal Essential Medium (MEM), Dulbecco'sModified Eagle Medium (DMEM), all of which may be supplemented withserum and/or growth factors as required by the particular cell linebeing cultured. A suitable medium for insect cultures is Grace's mediumsupplemented with yeastolate. lactalbumin hydrolysate, and/or fetal calfserum as necessary.

Typically, an antibiotic or other compound useful for selective growthof transfected or transformed cells is added as a supplement to themedia. The compound to be used will be dictated by the selectable markerelement present on the plasmid with which the host cell was transfonned.For example, where the selectable marker element is kanamycinresistance, the compound added to the culture medium will be kanamycin.Other compounds for selective growth include ampicillin. tetracyclineand neomycin

The amount of tmst2-receptor polypeptidc produced by a host cell can beevaluated using standard methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays.

If a tmst2-receptor polypeptide has been designed to be secreted fromthe host cells, the majority of polypeptide may be foulid in the cellculture medium. If however, the tmst2-receptor polypeptide is notsecreted from the host cells, it will be present in the cytoplasm and/orthe nucleus (for eukaiyotic host cells) or in the cytosol (for bacterialhost cells).

For a tmst2-receptor polypeptide situated in the host cell cytoplasmand/or nucleus, (for eukaryotic host cells) or in the cytosol (forbacterial host cells), intracellular material (including inclusionbodies for gram-negative bacteria) can be extracted from the host cellusing any standard technique known to the skilled artisan. For example,the host cells can be lysed to release the contents of theperiplasm/cytoplasm by French press, homogenization, and/or sonicationfollowed by centrifugation.

Purification of a tmst2-receptor polypeptide from solution can beaccomplished using a variety of tecmiques. If the polypeptidc has beensynthesized such that it contains a tag such as Hexahistidine(tmst2-receptor polypeptide/hexaHis) or other small peptide such as FLAG(Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen, Carlsbad,Calif.) or the IgG Fc fragment fused at either its carboxyl or aminoterminus, it may essentially be purified in a one-step process bypassing the solution through an affinity column where the column matrixhas a high affinity for the tag or for the polypeptide directly (i.e., amonoclonal antibody specifically recognizing tmst2-receptorpolypeptide). For example, polyhistidine binds with great affinity andspecificity to nickel, thus an affinity column of nickel (such as theQIAGEN® nickel columns) can be used for purification of tmst2-receptorpolypeptide/polyHis. (See for example, Ausubel et al. eds., CurrentProtocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, NewYork (1993)).

Where a tmst2-receptor polypeptide is prepared without a tag attached,and no antibodies are available, other well known procedures forpurification can be used. Such procedures include, without limitation,ion exchange chromatography, molecular sieve chromatography, HPLC,native gel electrophoresis in combination with gel elution, andpreparative isoelectric focusing (“Isoprime” machinc/technique, HoeferScientific). In some cases, two or more of these techniques may becombined to achieve increased purity.

If a tmst2-receptor polypeptide is produced intracellularly, theintracellular material (including inclusion bodies for gram-negativebacteria) can be extracted from the host cell using any standardtechnique known to the skilled artisan. For example, the host cells canbe lysed to release the contents of the periplasm/cytoplasm by Frenchpress, homogenization, and/or sonication followed by centrifugation.

If a tmst2-receptor polypeptide has formed inclusion bodies in thecytosol, the inclusion bodies can often bind to the inner and/or outercellular membranes and thus will be found primarily in the pelletmaterial after centrifugation. The pellet material can then be treatedat pH extremes or with chaotropic agent such as a detergent, guanidine,guanidinie derivatives, urea, or urea derivatives in the presence of areducing agent such as dithiothreitol at alkaline pH or triscarboxyethyl phosphine at acid pH to release, break apart, andsolubilize the inclusion bodies. The tmst2-receptor polypeptide in itsnow soluble form can then be analyzed using gel electrophoresis,immunoprecipitation or the like. If it is desired to isolate thetmst2-receptor polypeptide, isolation may be accomplished using standardmethods described herein below and in Marston et al. (Meth. Enz.,182:264-275(1990)).

In some cases, a tmst2-receptor polypeptide may not be biologicallyactive upon isolation. Various methods for “refolding” or converting thepolypeptide to its tertiary structure and generating disulfide linkages,can be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotope is used at a lowerconcentration and is not necessarily the same chaotropes as used for theSolubilizationi. In most cases the refolding/oxidatioln solution willalso contain a reducing agent or the reducing agent plus its' oxidizedform in a specific ratio to generate a particular redox potentialallowing for disulfide shufflin(g to occur in the formation of theprotein's cysteine bridge(s). Some of the commonly used redox couplesinclude cysteine/cystamine, gilutathione (GSH)/dithiobis GSH, cupricchloride, ditliothreitol(DTT)/ditlianie DTT,2-mercaptoethanol(βME)/dithio-β(ME). A cosolvent may be used to increasethe efficiency of the refolding and the more common reagents used forthis purpose include glycerol, polyethylene glycol of various molecularweights, arginine and the like.

If inclusion bodies are not formed to a significant degree uponexpression of a tmst2-receptor polypeptide, then the polypeptide will befound primarily in the supernatant after centrifugation of the cellhomogenate. The polypeptide may be further isolated from the supernatantusing methods such as those described herein.

Additionally, the tmst2-receptor polypeptide may be purified through theuse of a monoclonal antibody which is capable of specificallyrecognizing and binding to the tmst2-receptor polypeptide.

Suitable procedures for purification thus include, without limitation,affinity chromatography, immunoaffinity chromatography, ion exchangechromatography, molecular sieve chromatography, High Performance LiquidChromatography (HPLC), electrophoresis (including native gelelectrophoresis) followed by gel elution, and preparative isoelectricfocusing (“Isoprime” machine/technique, Hoefer Scientific, SanFrancisco, Calif.). In some cases, two or more purification techniquesmay be combined to achieve increased purity.

Tmst2-receptor polypeptides, fragments, and/or derivatives thereof mayalso be prepared by chemical synthesis methods (such as solid phasepeptide synthesis) using techniques known in the art such as those setforth by Merrifield et al., (J. Am. Chem. Soc., 85:2149 (1963)),Houghten et al. (Proc Natl Acat. Sci. USA, 82:5132 (1985)), and Stewartand Young (Solid Phase Peptide Sythesis, Pierce Chemical Co., Rockford,Ill. (1984)). Such polypeptidcs may be synthesized with or without amethionine on the amino terminus. Chemically synthesized tmst2-receptorpolypeptides or fragments may be oxidized using methods set forth inthese references to form disulfide bridges. Chemically synthesizedtmst2-receptor polypeptides, fragments or derivatives are expected tohave comparable biological activity to the corresponding tmst2-receptorpolypeptides, fragments or derivatives produced recombinanitly orpurified from natural sources, and thus may be used interchangeably withrecombinant or natural tmst2-receptor polypeptide.

Another means of obtaining tmst2-receptor polypeptide is viapurification from biological samples such as source tissues and/orfluids in which the tmst2-receptor polypeptide is naturally found. Suchpurification can be conducted using methods for. protein purification asdescribed herein. The presence of the tmst2-receptor polypeptide duringpurification may be monitored using, for example, an antibody preparedagainst recombinantly produced tmst2-receptor polypeptide or peptidefragments thereof.

A number of additional methods for producing nucleic acids andpolypeptides are known in the art, and can be used to producepolypeptides having specificity for tmst2 like. See for example, Robertset al., Proc. Natl. Acad. Sci. U.S.A., 94:12297-12303 (1997), whichdescribes the production of fusion proteins between an mRNA and itsencoded peptide. See also Roberts, Curr. Opin. Chem. Biol., 3:268-273(1999). Additionally, U.S. Pat. No. 5,824,469 describes methods ofobtaining oligonucleotides capable of carrying out a specific biologicalfunction. The procedure involves generating a heterogeneous pool ofoligonucleotides, each having a 5′ randomized sequence, a centralpreselected sequence, and a 3′ randomized sequence. The resultingheterogeneous pool is introduced into a population of cells that do notexhibit the desired biological function. Subpopulations of the cells arethen screened for those which exhibit a predetermined biologicalfunction. From that subpopulation, oligonucleotides capable of carryingout the desired biological function are isolated.

U.S. Pat. Nos. 5,763,192, 5,814,476, 5,723,323, and 5,817,483 describeprocesses for producing peptides or polypeptides. This is done byproducing stochastic genes or fragments thereof, and then introducingthese genes into host cells which produce one or more proteins encodedby the stochastic genes. The host cells are then screened to identifythose clones producing peptides or polypeptides having the desiredactivity.

Another method for producing peptides or polypeptides is described inPCT/US98/20094 (WO99/15650) filed by Athersys, Inc. known as “RandomActivation of Gene Expression for Gene Discovery” (RAGE-GD), the processinvolves the activation of endogenous gene expression or over-expressionof a genc by in situ recombination methods. For example, expression ofan endogenous gene is activated or increased by integrating a regulatorysequence into the target cell which is capable of activating expressionof the gene by non-homologous or illegitimate recombination. The targetDNA is first subjected to radiation, and a genetic promoter inserted.The promoter eventually locates a break at the front of a gene,initiating transcription of the gene. This results in expression of thedesired peptidc or polypeptide.

It will be appreciated that these methods can also be used to createcomprehensive IL-17 like protein expression libraries, which cansubsequently be used for high throughput phenotypic screening in avariety of assavs, such as biochemical assays, cellular assays, andwhole organism assays (e.g., plant, mouse, etc.).

Proteins, Polypeptides, Fragments, Variants and Muteins of tmst2:

Polypeptides of the invention include isolated tmst2-receptorpolypeptides and polypeptides related thereto including fragments,variants, fusion polypeptides, and derivatives as defined herein above.

Tmst2-receptor fragments of the invention may result from truncations atthe amino terminus (with or without a leader sequence), truncations atthe carboxy terminus, and/or deletions internal to the polypeptide. Mostdeletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of thetmst2-receptor protein. However, when it is difficult to predict theexact effect of the substitution, deletion, or insertion in advance ofdoing so, one skilled in the art will appreciate that the effect will beevaluated by routine screening assays. For example, a variant typicallyis made by site-specific mutagenesis of the tmst2-receptorpolypeptide-encoding nucleic acid, expression of the variant nucleicacid in recombinant cell culture, and, optionally, purification from thecell culture, for example, by immunoaffinity adsorption on a polyclonalanti tmst2-receptor antibody column (to absorb the variant by binding itto at least one remaining immune epitope). In preferred embodiments,truncations and/or deletions comprise about 10 amino acids, or about 20amino acid, or about 50 amino acids, or about 75 amino acids, or about100 amino acids, or more than about 100 amino acids. The polypeptidefragments so produced will comprise about 25 contiguous amino acids, orabout 50 amino acids, or about 75 amino acids, or about 100 amino acids,or about 150 amino acids, or about 175 amino acids or 190 amino acids.

Tmst2-receptor polypeptide variants of the invention include one or moreamino acid substitutions, additions and/or deletions as compared to SEQID NO: 8. In preferred embodiments, the variants have from 1 to 3, orfrom 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or morethan 100 amino acid substitutions, insertions, additions and/ordeletions, wherein the substitutions may be conservative, as definedabove, or non-conservative or any combination thereof. More particularlytmst2-receptor variants may comprise the amino acid sequence set out asSEQ ID NO: 8, wherein one or more amino acids from the group consistingof amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, and 198 is substituted withanother amino acid.

Tmst2-receptor-receptor variants may also comprise the amino acidsequence set out as SEQ ID NO.: 8, wherein one or more amino acids fromthe group consisting ofamino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 14, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, and 198is/are deleted,

Receptor variants of the secreted form of the tmst-2 receptor (tmst2)are also envisioned within the scope of the invention. Variants of thesecreted form of the tmst2-receptor may comprise the amino acid sequenceset out as SEQ ID NO: 10, wherein one or more amino acids from the groupconsisting of amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, and 180 is/are substituted with anotheramino acid.

Soluble tmst2-receptor variants may also comprise the amino acidsequence set out as SEQ ID NO: 10, wherein one or more amino acids fromthe group consisting of amino acids 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, and 180 is/are deleted,

The variants may also lhave additions of amino acid residues either atthe carboxy terminus or at the amino terminus (with or without a leadersequence).

Preferred tmst2-rcccptor polypeptide variants include glycosylationvariants wherein the number and/oor type of glycosylation sites has beenaltered compared to native tmst2-receptor polypeptide. In oneembodiment, tmst2-receptor variants comprise a greater or a lessernumber of N-linked glycosylation sites. An N-linked glycosylation siteis characterized by the sequence: Asn-X-Ser or Thr, where the amino acidresidue designated as X may be any type of amino acid except proline.Substitution(s) of amino acid residues to create this sequence providesa potential new site for addition of an N-liniked carbohydrate chain.Alternatively, substitutions to eliminate this sequence will remove anexisting N-linked carbohydrate chain. Also provided is a rearrangementof N-linked carbohydrate chains wherein one or more N-linkedglycosylation sites (typically those that are naturally occurring) areeliminated and one or more new N-linked sites are created.

One skilled in the art will be able to determine suitable variants ofthenative tmst2-receptor polypeptide using well known techniques. Forexample, one may be able to predict suitable areas ofthe molecule thatmay be changed without destroying biological activity. Also, one skilledin the art will realize that even areas that may be important forbiological activity or for stnIcture may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the polypeptide structure.

For predicting suitable areas of the molecule that may be changedwithout destroying activity, one skilled in the art may target areas notbelieved to be important for activity. For example, when similarpolypeptides with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of tmst2-receptor polypeptide to such similar polypeptides.After making such a comparison, one skilled in the art would be able todetermine residues and portions of the molecules that are conservedamong similar polypeptides. One skilled in the art would know thatchanges in areas of the tmst2-receptor molecule that are not conservedwould be less likely to adversely affect biological activity and/orstricture. One skilled in the art would also know that, even inrelatively conserved regions, one could have likely substitutedchemically similar amino acids for the naturally occurring residueswhile retaining activity (e.g. conservative amino acid residuesubstitutions).

Also, one skilled in the art may review strmcture-function studiesidentifying residues in similar polypeptides that are important foractivity or structure. In view of such a comparison, one skilled in theart can predict the importance of amino acid residues in tmst2-receptorthat correspond to amino acid residues that are important for activityor structure in similar polypeptides. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues of tmst2-receptor.

If available, one skilled in the art can also analyze the crystalstructure and amino acid sequence in relation to that structure insimilar polypeptides. In view of that infonnation, one skilled in theart may be able to-predict the alignment of amino acid residues oftmst2-receptor polypeptide with respect to its three dimensionalstructure. One skilled in the art may choose not to make radical changesto amino acid residues. predicted to be on the surface of the protein,since such residues may be involved in important interactions with othermolecules.

Moreover, one skilled in the art could generate test variants containinga single amino acid substitution at each amino acid residue. Thevariants could be screened using activity assays disclosed in thisapplication. Such variants could be used to gather information aboutsuitable variants. For example, if one discovered that a change to aparticular amino acid residue resulted in destroyed activity, variantswith such a change would be avoided. In other words, based oninformation gathered from such experiments, when attempting to findadditional acceptable variants, one skilled in the art would have knownthe amino acids where further substitutions should be avoided eitheralone or in combination with other mutations.

Tmst2-receptor fusion polypeptides of the invention comprisetmst2-receptor polypeptides, fragments, variants, or derivatives fusedto a heterologous peptide(s) or protein(s). Heterologous peptide(s) andprotein(s) include, but are not limited to, an epitope to allow fordetection and/or isolation of a tmst2-receptor fusion polypeptide, atransmembrane receptor protein or a portion thereof, such as anextracellular domain, or a transmembrane, a ligand or a portion thereofwhich binds to a transmembrane receptor protein, an enzyme or portionthereof which is catalytically active, a protein or peptide whichpromotes oligomerization, such as leucine zipper domain, and a proteinor peptide which increase stability, such as an immunoglobulin constantregion. A tmst2-receptor polypeptide may be fused to itself or to afragment, variant. or derivative thereof. Fusions may be made either atthe amino terminus or at the carboxy terminus of a tmst2-receptorpolypeptide, and may be direct with no linker or adapter molecule or maybe through a linker or adapter molecule. such as one or more amino acidresidues up to about 20 amino acids residues, or up to about 50 aminoacid residues. Altcnatively, the tmst2-receptor fusion protein maycomprise one or two tmst2-receptor polypeptides covalently linked to oneor two TNF-receptor polypeptide(s), or a member of the TNF-receptorfamily or a cytokinc receptor such as interleukin-1 R (IL-1R)polypeptide. The receptors preferably are produced as fusion proteinsusing recombinant DNA technology. A linker or adapter molecule may alsobe designed with a cleavage site for a DNA restriction endonuclease orfor proteolytic cleavage to allow for separation and subsequent foldingof the fuised moieties.

Also envisioned as a part of the invention are circularly permutedstructural analogs of the tmst2-receptor polypeptide.

The development of recombinant DNA methods has made it possible to studythe effects of sequence transposition on protein folding, structure andfunction. The approach used in creating new sequences resembles that ofnaturally occurring pairs of proteins that are related by linearreorganization of their amino acid sequences (Cunningham, et al., Proc.Natl. Acad. Sci. U.S.A. 76:3218-3222, (1979); Teather & Erfle, J.Bacteriol. 172:3837-3841, (1990); Schimming et al., Eur. J. Biochlem.204:13-19, (1992); Yamiuchi and Minamikawa, FEBS Lett 260:127-1 30,(1991); MacGregor et al., FEBS Lett. 378:263-266, (1996)). The first invitro application of this type of rearrangement to proteins wasdescribed by Goldenberg and Creighton (J. Mol. Biol. 165:407-413,(1983)). A new N-temiinus is selected at an internal site (breakpoint)of the original sequence, the new sequence having the same order ofamino acids as the original from the breakpoint until it reaches anamino acid that is at or near the original C-terminus. At this point thenew sequence is joined, either directly or through an additional portionof sequence (linker), to an amino acid that is at or near the originalN-terminus, and the new sequence continues with the same sequence as theoriginal until it reaches a point that is at or near the amino acid thatwas N-terminal to the breakpoint site of the original sequence, thisresidue forming the new C-terminus of the chain.

This approach has been applied to proteins which range in size from 58to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol. 165:407-413,(19830; Li & Coffino, Mol. Cell. Biol. 13:2377-2383, (1993)). Theproteins examined have represented a broad range of structural classes,including proteins that contain predominantly a α-helix (interleukin-4;Kreitman et al., Cytokine 7:311-318, (1995)), β-sheet (interieukin-1;Horlick et al., Protein Eng. 5:427-431, (1992)), or mixtures of the two(yeast phosphoribosyl anthranilate isomerase; Luger et al., Science243:206-210, (1989)).

In a preferred embodiment, a tmst2-receptor polypeptide, fragment,variant and/or derivative is fused to an Fc region of human IgG. In oneexample, a human IgG hinge, CH2 and CH3 region may be fused at eitherthe N-teniinus or C-terminus of the tmst2-reccptor polypeptides usingmethods known to the skilled artisan. In another example, a portion of ahinge regions and CH2 and CH3 regions may be fuse. The tmst2-receptor Fc-fusion polypeptide so produce may be purified by use of a Protein Aaffinity column (Pierce, Rockford, Ill.). In addition, peptide andproteins fused to an Fc region have been found to exhibit asubstantially greater half-life in vivo than the unfused counterpart.Also, a fusion to an Fc region allows for dimerization/multimerizationof the fusion polypeptide. The Fc region may be naturally occurring Fcregion, or may be altered to improve certain qualities such astherapeutic qualities, circulation time, reduce aggregation, etc.

Tmst2-receptor-receptor polypeptide derivatives are also included in thescope of the present invention. Covalent modifications of thetmst2-receptor proteins of the present invention are included within thescope of this invention. Variant tmst2-receptor proteins maybeconveniently prepared by in vitto synthesis. Such modifications may beintroduced into the molecule by reacting targeted amino acid residues ofthe purified or crzde protein with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues. Theresulting covalent derivatives are useful in programs directed atidentifying residues important for biological activity.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), Such as chloroacetic acid or chloroacetamide, togive carboxyiethyl or carbocyamidomiiethyl derivatives. Cysteinylresidues also are derivatized by reaction with bromotri fLuoroacetonie,α-bromo-β(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmalcimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic orcarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineEpsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod described above being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R¹) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpenityl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking the“tmst2-receptor proteins” to water-insoluble support matrixes orsurfaces for use in the method for cleaving the tmst2-receptorprotein-fusion polypeptide to release and recover the cleavedpolypeptide. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctionial imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimnides such as bis-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming cross links in the presence oflight. Alteniatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440, incorporated herein by reference, are employed forprotein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure find MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups. Such derivatives are chemicallymodified tmst2-receptor polypeptide compositions in which tmst2-receptorpolypeptide is linked to a polymer. The polymer selected is typicallywater soluble so that the protein to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. The polymer selected is usually modified to have a singlereactive group, such as an activc ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled asprovided for in the present methods. Fhe polymer may be of any molecularweight, and may be branched or unbranched. Icluided within the scope ofthe tmst2-receptor polypeptide polymers is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of, for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol.

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. A preferredreactive aldehyde is polyethylene glycol propionaldehyde, which is waterstable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S.Pat. No. 5,252,714).

Pegylation of tmst2-receptor polypeptides may be carnied out by any ofthe pegylation reactions known in the art, as described for example inthe following references: Focus on Growth Factors 3: 4-10 (1992); EP 0154 316; and EP 0 401 384 incorporated herein by reference. Preferably,the pegylation is carried out via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule (or an analogousreactive water-soluble polymer) as described below.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol. PEG is a linear or branched neutralpolyether, available in a broad range of molecular weights, and issoluble in water and most organic solvents. PEG is effective atexcluding other polymers or peptides when present in water, primarilythrough its high dynamic chain mobility and hydrophibic nature, thuscreating a water shell or hydration sphere when attached to otherproteins or polymer surfaces. PEG is nontoxic, non-immunogenic, andapproved by the Food and Drug Administration for internal consumption.

Proteins or enzymes when conjugated to PEG have demonstratedbioactivity, non-anttigenic properties, and decreased clearance rateswhen administered in animals. F. M. Veronese et al., Preparation andProperties of Monomethoxypoly(ethyene glyco.)-modified Enzymes forTheraeutic Applications, in J. M. Harris ed., Poly(Ethylene Clycol)Chemistry—Biotechnical and Biomedical Applications 127-36 (1 992),incorporated herein by reference. This is due to the exclusionproperties of PEG in preventing recognition by the immune system. Inaddition, PEG has been widely used in surface modification procedures todecrease protein adsorption and improve blood compatibility. Kim et al.,Ann. N. Y. Acad. Sci. 516:116-30 (1987); Jacobs et al., Artif. Organs12:500-01 (1988); Park et al., J. Poly. Sci, Part A 29:1725-31 (1991),incorporated herein by reference. Hydrophobic polymer surfaces, such aspolyurethanes and polystyrene were modified by the grafting of PEG (MW3,400) and employed as nonthrombogenic surfaces. In these studies,surface properties (contact angle) were more consistent with hydrophilicsurfaces. due to the hydrating effect of PEG. More importantly, protein(albumin and other plasma proteins) adsorption was greatly reduced,resulting from the high chain motility, hydration sphere, and proteinexclusion properties of PEG.

PEG (MW 3,4000) was deternined as an optimal size in surfaceimmobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45(1992), while PEG (MW 5,000) was most beneficial in decreasing proteinantigenicity. (Veronese et al., In Harris el al., Poly(Ethylene Glycol)Chemistry—Biotechnical and Biotechnical Applications 127-36, supra.,incorporated herein by reference)

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylatedtmst2-receptor polypeptides will generally comprise the steps of (a)reacting the polypeptide with polyethylene glycol (such as a reactiveester or aldehyde derivative of PEG) under conditions wherebytmst2-receptor polypeptide becomes attached to one or more PEG groups,and (b) obtaining the reaction product(s). In general, the optimalreaction conditions for the acylation reactions will be determined basedon known parameters and the desired result. For example, the larger theratio of PEG: protein, the greatcr the percentage of poly-pegylatedproduct.

In a preferred embodiment, the tmst2-receptor polypeptide derivativewill have a single PEG moiety at the N terminus. See U.S. Pat. No.8,234,784, herein incorporated by reference.

In another embodiment, tmst2-receptor polypeptides may be chemicallycoupled to biotin, and the biotin/tmst2 like polypeptide molecules whichare cotjugatedc are then allowed to bind to avidin, resulting intetravalent avidinibiotin/tmst2 like polypeptide molecules. Tmst2 likepolypeptides may also be covalently coupled to dinitrophenol (DNP) ortrinitropheniol (TNP) and the resulting conjugates precipitated withanti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of10.

Generally, conditions which may be alleviated or modulated byadministration of the present tmst2-receptor polypeptide derivativeinclude those described herein for tmst2-receptor polypeptides. However,the tmst2-reccptor polypeptide derivative disclosed herein may haveadditional activities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

Selective Binding Agents

As used herein, the term “selective binding agent” refers to a moleculewhich has specificity for one or more tmst2-receptor polypeptides.Suitable selective binding agents include, but are not limited to,antibodies and derivatives thereof, polypeptides, antisenseoligonucleotides, and small molecules. Suitable selective binding agentsmay be prepared using methods known in the art. An exemplarytmst2-receptor polypeptidc selective binding agent of the presentinvention is capable of binding a certain portion of the tmst2 likepolypeptide thereby inhibiting the binding of the polypeptide to thetmst2 like polypeptide receptor(s).

Selective binding agents such as antibodies and antibody fragments thatbind tmst2-receptor polypeptides are within the scope of the presentinvention. The antibodies may be polyclonal including monospecificpolyclonal, monoclonal (MAbs), recombinant, chimeric, humanized such asCDR-,rafted, human, single chain, and/or bispecific, as well asfragments, variants or derivatives thereof. Antibody fragmcnts includethose portions of the antibody which bind to an epitope on the tmst2like polypeptide. Examples of such fragments include Fab and F(ab′)fragments generated by enzymatic cleavage of full-length antibodies.Other binding fragments include those generated by recombinant DNAtechniques, such as the expression of recombinant plasmids containingnucleic acid sequences encoding antibody variable regions.

Polyclonal antibodies directed toward a tmst2-receptor polypeptidegenerally are produced in animals (e.g., rabbits or mice) by means ofmultiple subcutaneous or intraperitoneal injections of tmst2-receptorand an adjuvant. It may be useful to conjugate a tmst2-receptorpolypeptide, or a variant, fragment or derivative thereof to a carrierprotein that is immunogenic in the species to be immrunized, such askeyhole limpet heocyanin, serum, albumin, bovine thyroglobulin, orsoybean trypsin inhibitor. Also, aggregating agents such as alum areused to enhance the immune response. After immunization, the animals arebled and the serum is assayed for anti-tmst2-receptor antibody titer.

Monoclonal antibodies directed toward tmst2-receptor polypeptides areproduced using any method which provides for the production of antibodymolecules by continuous cell lines in culture. Examples of suitablemethods for preparing monoclonal antibodies include the hybridomamethods of Kohler, et al., Nature, 256: 495-497 (1975), and the humanB-cell hybridoma method, Kozbor, J. Immunol., 133: 3001 (1984); Brodeur,et al., Monoclonal Atitibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987).

Also provided by the invention are hybridoma cell lines which producemonoclonal antibodies reactive with tmst2-receptor polypeptides.

Monoclonal antibodies of the invention may be modified for use astherapeutics. One embodiment is a “chimeric” antibody in which a portionof the heavy and/or light chain is identical with or homologous tocorresponding sequence in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequence in antibodies derived from another species orbelonging to another antibody class or subclass. As included arefragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; Morrison, et al.,Proc. Natl. Acad. Sci. U.S.A. 81,6851-6855 (1985)).

In another embodiment, a monoclonal antibody of the invention is a“humanized” antibody. Methods for humanizing non-human antibodies arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.Humanization can be performed following methods known in the art (Joneset al., Nature, 321: 522-525 (1986); Riechmann, et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), bysubstituting rodent complementarity-determiniing regions (CDRs) for thecorresponding regions of a human antibody.

Also encompassed by the invention are fully human antibodies which bindtmst2-receptor polypeptides, fragments, variants and/or derivatives.Using transgenic animals (e.g., mice) that are capable of producing arepertoire of human antibodies in the absence of endogenousimmunoglobulin production such antibodies are produced by immunizingwith a tmst2-receptor antigen (i.e., having at least 6 contiguous aminoacids), optionally conjugated to a carrier. See, for example, Jakobovitset al., Proc. Natl. Acid. Sci. U.S.A., 90: 2551-2555 (1993); Jakobovitset al., Nature, 362: 255-258 (1993); Bruggermann et al., Year inImmuno., 7:33 (1993). In one method, such transgenic animals areproduced by incapacitating the endogenous loci encoding the heavy andlight immtnoglobulin chains therein, and inserting loci encoding humanheavy and light chain proteins into the genome thereof. Partiallymodified animals, that is those having less than the full complement ofmodifications, are then cross-bred to obtain an animal having all of thedesired immune system modifications. When administered an immunogen,these transgenic animals produce antibodies with human (rather thane.g., murine) amino acid sequences, including variable regions which areimmunospecific for these antigens. Sec PCT Application Nos.PCT/US96/05928 and PCT/US93/06926. Additional methods are described inU.S. Pat. No. 5,545,807, PCT application Nos. PCT/US91/245,PCT/GB89/01207, and in EP 546073B1 and EP 546073A1. Human antibodies mayalso be produced by the expression of recombinant DNA in host cells orby expression in hybridoma cells as described herein.

In an alternate embodiment, human antibodies can also be produced inphage-display libraries (Hoogenboom, el al., J. Mol. 1 Biol. 227:381(1991); Marks, el al., J. Mol. Biol. 222:581 (1991). These processesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in PCT Application No. PCT/US98/17364, which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

Chimeric, CDR grafted, and humanized antibodies are typically producedby recombinant methods. Nucleic acids encoding the antibodies areintroduced into host cells and expressed using materials. and proceduresdescribed herein. In a preferred embodiment, the antibodies are producedin mammalian host cells, such as CHO cells. Monoclonal (e.g., human)antibodies may be produced by the expression of recombinant DNA in hostcells or by expression in hybridoma cells as described herein.

For diagnostic applications, anti-tmst2-receptor antibodies typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,β-galactosidase or horseradish peroxidase. Bayer et al., Meth. Enz.,184: 138-163 (1990).

The anti-tmst2-receptor antibodies of the invention may be employed inany known assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (Sola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc., 1987)) for the detection and quantitation of tmst2-receptorpolypeptides. The antibodies will bind tmst2-receptor polypeptides withan affinity which is appropriate for the assay method being employed.

The activity of the cell lysate or puri Fied tmst2 receptor proteinvariant is then screened in a suitable screening assay for the desiredcharacteristic. For example, a change in the binding affinity for aligand or immunological character of the tmst2 receptor protein, such asaffinity for a given antibody, is measured by a competitive typeimmunoassay. Changes in immuomodulation activity are measured by theappropriate assay. Modifications of such protein properties as redox orthermal stability hydrophobicity, susceptibility to proteolyticdegradation or the tendency to aggregate with carriers or into multimersare assayed by methods well known to the ordinarily skilled artisan.Competitive binding assays rely on the ability of a labeled standard(e.g., a tmst2-receptor polypeptide, or an immunologically reactiveportion thereof) to compete with the test sample analyte (atmst2-receptor polypeptide) for binding with a limited amount ofantibody. The amount of a tmst2-receptor polypeptide in the test sampleis inversely proportional to the amount of standard that becomes boundto the antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies typically are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich imuno-assay typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is bound by a first antibody which is immobilized ona solid support, and thereafter a second antibody binds to the analyte,thus forming an insoluble three part complex. See e.g., U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbant assay (ELISA), in which case thedetectable moiety is an enzyme.

The anti-tmst2-receptor antibodies of the invention also are useful forin vivo imaging. An antibody labeled with a detectable moiety isadministered to an animal, preferably into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.The antibody may be labeled with any moiety that is detectable in ananimal, whether by nuclear magnetic resonance, radiology, or otherdetection means known in the art.

Selective binding agents of the invention, including anti-tmst2-receptorantibodies. may be used as therapeutics. These therapeutic antibodiesare generally agonists or antagonists, in that they either enhance orreduce, respectively, at least one or the biological activities of atmst2-receptor polypeptide. In one embodiment, antagonist antibodies ofthe invention are antibodies or binding fragments thereof which arecapable of specifically binding to a tmst2-reccptor polypeptide,fragment, variant and/or derivative, and which are capable of inhibitingor eliminating the functional activity of a tmst2-receptor polypeptidein vivo or in vitro. In preferred embodiments, an antagonist antibodywill inhibit the functional activity of a tmst2-receptor polypeptide atleast about 50%, preferably at least about 80%, more preferably 90%, andmost preferably 100%. Agonist and antagonist anti-tmst2-receptorantibodies are identified by screening assays described below.

Tmst2-receptor polypeptides can be used to clone tmst2-receptorligand(s) using an “expression cloning” strategy. Radiolabeled(¹²⁵-Iodine) tmst2-receptor polypeptide or “affinity/activity-tagged”tmst2-receptor like polypeptide (such as an Fc fusion or an alkalineplhosphatase fusion) can be used in binding assays to identify a celltype or a cell line or tissue that expresses tmst2-receptor ligand(s).RNA isolated from such cells or tissues can then be converted to cDNA,cloned into a mammalian expression vector, and transfected intomammalian cells (for example, COS, or 293) to create an expressionlibrary. Radiolabeled or tagged tmst2-receptor polypeptide can then beused as an affinity reagent to identify and isolate the subset of cellsin this library expressing tmst2-receptor ligand(s). DNA is thenisolated from these cells and transfected into mammalian cells to createa secondary expression library in which the fraction of cells expressingtmst2-receptor ligand(s) would be many-fold higher than in the originallibrary. This enrichment process can be repeated iteratively until asingle recombinant clone containing an tmst2-receptor ligand isisolated. Isolation of tmst2-receptor ligand(s) is useful foridentifying or developing novel agonists and antagonists of thetmst2-receptor signaling pathway. Such agonists and antagonists includetmst2-receptor ligand(s), anti-tmst2-receptor ligand antibodies, smallmolecules or antisense oligonucleotides.

Diagnostic Kits and Reagents

This invention also contemplates use of tmst2-receptor proteins,fragments thereof, peptides, binding compositions, and their fusionproducts in a variety of diaginostic kits and methods for detecting thepresence of receptors and/or antibodies, or ligands. Typically the kitwill have a compartment containing a tmst2-receptor peptide or genesegment or a reagent which recognizes one or the other, e.g., bindingreagents.

A kit for determining the binding affinity of a ligand or test compoundto the tmst2-receptor would typically comprise a test compound; alabeled compound, for example an antibody having known binding affinityfor the protein; or a source of ligand (naturally occurring orrecombinant), and a means for separating bound from free labeledcompound, such as a solid phase for immobilizing the ligand or receptor.Once compounds are screened, those having suitable binding affinity tothe ligand or receptor can be evaluated in suitable biological assays,as are well known in the art, to determine whether they act as agonistsor antagonists to the receptor. The availability of recombinantchemokine or receptor polypeptides also provide well defined standardsfor calibrating such assays or as positive control samples.

A preferred kit for determining the concentration of, for example,tmst2-receptor or ligand in a sample would typically comprise a labeledcompound, e.g., antibody, having known binding affinity for the target,a source of ligand or receptor (naturally occurring or recombinant), anda means for separating the bound from free labeled compound, forexample, a solid phase for immobilizing the ligand or receptor.Compartments containing reagents, and instructions for use or disposal,will normally be provided.

Antibodies, including antigen binding fragments, specific for the ligandor receptor, or fragments are useful in diagnostic applications todetect the presence of elevated levels of ligand, receptor, and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescenice, cell cultures, body fluids, and further caninvolve the detection of antigens related to the ligand or receptor inserum, or the like. Diagnostic assays may be homogeneous (without aseparation step between free reagent and antigen complex) orheterogeneous (with a separation step). Various commercial assays exist,such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassaytechnique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), andthe like. For example, unlabeled antibodies can be employed by using asecond antibody which is labeled and which recognizes the primaryantibody to a ligand or receptor or to a particular fragment thereof.Similar assays have also been extensively discussed in the literature.(See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, CSH, 1988)

Anti-idiotypic antibodies may have similar uses to diagnose presence ofantibodies against a chemokine or receptor, as such may be diagnostic ofvarious abnormal states. For example, overproduction of a chemokine orreceptor may result in production of various immunological reactionswhich may be diagnostic of abnormal physiological states, particularlyin various inflammatory or asthma conditions.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or labeled chemokine or receptor isprovided. This is usually in conjunction with other additives, such asbuffers, stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments or containers for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

The aforementioned constituents of the drug screening and the diagnosticassays may be used without modification or may be modified in a varietyof ways. For example, labeling may be achieved by covalently ornon-covalently joining a moiety which directly or indirectly provides adetectable signal. In any of these assays, the ligand, test compound,receptor, or antibodies thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵I, enzyines (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating bound from the freeligand, or alternatively bound from free test compound. The chemokine orreceptor can be immobilized on various matrixes, perhaps with detergentsor associated lipids, followed by washing. Suitable matrixes includeplastic such as an ELISA plate, filters, and beads. Methods ofimmobilizing the chemokine or receptor to a matrix include, withoutlimitation, direct adhesion to plastic, use of a capture antibody,chemical coupling, and biotin-avidin. The last step in this approach mayinvolve the precipitation of antigen/antibody complex by any of severalmethods including those utilizing, e.g., an organic solvent such aspolyethylene glycol or a salt such as ammonium sulfate. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (Clin.Chem.,30:1457-1461 (1984)), and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,6178, incorporated hereinby reference.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Nucleic acid molecules of the invention may be used to map the locationsof the tmst2-receptor gene and related genes on chromosomes. Mapping maybe done by techniques known in the art, such as PCR amplification, insitu hybridization, and FISH.

This invention is also related to the use of the tmst2-receptor gene aspart of a diagnostic assay for detecting diseases or susceptibility todiseases related to the presence of mutated tmst2-receptor gene. Suchdiseases are related to an abnormal expression of tmst2-receptor, forexample, abnonnal cellular proliferation such as tumors and cancers.

Individuals carrying mutations in the human tmst2-receptor gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The gcnomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding tmst2-receptorpolypeptide can be used to identify and analyze tmst2 receptormutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled tmst2 receptor RNA or alternatively radiolabeled tmst2receptor antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length POLYMORPPHISMS (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of tmst2-receptor protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, tumors, cerebral malaria and hereditary periodicfever syndromes. Assays used to detect levels of tmst2-receptor proteinin a sample derived from a host are well-known to those of skill in theart and include radioimmunoassays, competitive-binding assays, WesternBlot analysis, ELISA assays and “sandwich” assay. An ELISA assay(Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6,(1991)) partially comprises preparing an antibody specific to thetmst2-receptor antigen, preferably a monoclonal antibody. In addition areporter antibody is prepared against the monoclonal antibody. To thereporter antibody is attached a detectable reagent such asradioactivity, fluorescence or in this example a horseradish peroxidaseenzyme. A sample is now removed from a host and incubated on a solidsupport, e.g., a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like BSA. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attach to any tmst2-receptor proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to tmst2-receptor. Unattached reporterantibodyis then washed out. Peroxidase substrates are then added to thedish and the amount of color developed in a given time period is ameasurement of the amount of tmst2-receptor protein present in a givenvolume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to tmst2receptor are attached to a solid support and labeled tmst2-reccptor anda sample derived from the host are passed over the solid support and theamount of label detected, for example, by liquid scintillationchromotagraphy, can be correlated to a quantity of tmst2 receptor in thesample. In addition, a “sandwich” immuno-assay as described above mayalso be carried out to quantify the amount of tmst2-receptor in abiological sample.

The sequences ofthe present invention are also valuable for chromosomeidentification and mapping. The sequence can be specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying,particular sites on the chromosome wherein a gene can be localized. Fewchromosome marking reagents based on actual sequence data (repeatPOLYMORPHISMS) are presently available for marking chromosomal location.The mapping of DNAs to chromosomes according to the present invention isan important first step in correlating those sequences with genesassociated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCRprimiers (preferably 15-25 bp) from the cDNA. Computer analysis of the3′-untranslated region of the sequence is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map tmst2-receptor to its chromosome include insitu hybridization, prescreening with labeled flow-sorted chromosomesand preselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of genomic clones orclones from which the express sequence tag (EST) was derived, and thelonger the better. For example, 2,000 bp is good, 4,000 is better, andmore than 4,000 is probably not necessary to get good results areasonable percentage of the time. For a review of this technique seeVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals. then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be on of between 50 and 500 potentialcausative geties (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The nucleic acid molecule(s) of the present invention are also used asanti-sense inhibitors of tmst2 receptor expression. Such inhibition maybe effected by nucleic acid molecules which are complementary to andhybridize to expression control sequences (triple helix fonnation) or totmst2-receptor miNIA. Anti-sense probes may be designed by availabletechniques using the sequence of tmst2-receptor disclosed herein.Anti-sense inhibitors provide informnation relating to the decrease orabsence of a tmst2-receptor polypeptide in a cell or organism. Thenucleic acid molecules of the invention may be used for gene therapy.Nucleic acid molecules which express tmst2-receptor in vivo provideinformation relating to the effects of the polypeptide in cells ororganisms. Tmst2-receptor nucleic acid molecules, fragments, and/orderivatives that do not themselves encode biologically activepolypeptides may be useful as hybridization probes in diagnostic assaysto test, either qualitatively or quantitatively, for the presence oftmst2 receptor DNA or corresponding RNA in mammalian tissue or bodilyfluid samples.

Tmst2-receptor polypeptide fragments, variants, and/or derivatives,whether biologically active or not, are useful for preparing antibodiesthat bind to an tmst2-receptor polypeptide. The antibodies may be usedfor in vivo and in vitro diagnostic purposes, such as in labeled form todetect the presence of tmst2-receptor polypeptide in a body fluid orcell sample. The antibodies may bind to an tmst2-receptor polypeptide soas to diminish or block at least one activity characteristic of antnst2-receptor polypeptide, or may bind to a polypeptide to increase anactivity.

Genetically Engineered Non-Human Mammals

The present invention further includes non-human mammals such as mice,rats, rabbits, goats, or sheep in which the gene (or genes) encodingtmst2-receptor polypeptides in which either the native form of thegene(s) for that mammal or a heterologous tmst2-receptor polypeptidegene(s) is (are) over expressed by the mammal, thereby creating a“transgenic” mammal. Such transgenic mammals may be prepared using wellknown methods such as those described in U.S. Pat. No. 5,489,743 and PCTPublication No. W094/28 122, incorporated herein by reference.

Additionally included within the scope of the present invention arenon-human mammals such as mice, rats, rabbits, goats, or sheep in whichthe gene (or genes) encoding a native tmst2-receptor polypeptide has(have) been disrupted (“knocked out”) such that the level of expressionof this gene or genes is (are) significantly decreased or completelyabolished. Such mammals may be prepared using techniques and methodssuch as those described in U.S. Pat. No. 5,557,032, incorporated hereinby reference.

The present invention further includes non-human mammals in which thepromoter for one or more of the tmst2-receptor polypeptides of thepresent invention is either activated or inactivated (using homologousrecombination methods as described below) to alter the level ofexpression of one or more of the native tmst2-receptor polypeptides.

These non-human mammals may be used for drug candidate screening. Theimpact of a drug candidate on the mammal may be measured. For example,drug candidates may decrease or increase expression of thetmst2-receptor polypeptide gene. In certain embodiments, the amount oftmst2-receptor polypeptide or a fragment(s) that is produced may bemeasured after exposure of the mammal to the drug candidate. Addionally.certain embodiments, one may detect the actual impact of the drugcandidate on the animal. For example, over expression of a particulargene may result in, or be associated with, a disease or pathologicalcondition. in such cases, one may test a drug candidate's ability todecrease expression of the gene or its ability to prevent or inhibit apathological condition. In other examples, production of a particularmetabolic product such as a fragment of a polypeptide, may result in, orbe associated with, a disease or pathological condition. In such cases,one may test a drug candidate's ability to decrease production of such ametabolic product or its ability to prevent or inhibit a pathologicalcondition,

Microarray

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array has numerous copies of asingle species of DNA which acts as a target for hybridization for itscognate mRNA. In expression profiling using DNA microarray technology,mRNA is first extracted fronm a cell or tissue sample and then convertedenzyrnatically to fluorescently labeled cDNA. This material ishybridized to the microarray and unbound cDNA is removed by washing. Theexpression of discrete genes represented on the array is then visualizedby quantitating the amount of labeled cDNA which is specifically boundto each target DNA. In this way, the expression of thousands of genescan be quantitated in a high throughput, parallel manner from a singlesample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the tmst2-receptor molecules of theinvention, including, but not limited to: the identification andvalidation of tmst2-receptor disease-related genes as targets fortherapeutics; molecular toxicology of tmst2-receptor molecules andinhibitors thereof; stratification of populations and generation ofsurrogate markers for clinical trials; and enhancing tmst2-receptorrelated small molecule drug discovery by aiding in the identification ofselective compounds in high throughput screens (H TS).

Assaying for Other Modulators of tmst2-Receptor Polypeptide Activity

In some situzations, it may be desirable to identify molecules that aremodulators, i.e., agonists or antagonists, of the activity oftmst2-receptor polypeptide. Natural or synthetic molecules that modulatetmst2-receptor can be identified using one or more of the screeningassays described below. Such molecules may be administered either in anex vivo manner, or in an in vivo manner by local or iv injection, or byoral delivery, implantation device, or the like.

The following definition is used herein for describing the assays:

“Test molecule(s)” refers to the molccule(s) that is/are underevaluation for the ability to modulates i.e., increase or decrease) theactivity of an tmst2-receptor polypeptide. Most commonly, a testmolecule will interact directly with an tmst2-receptor polypeptide.However, it is also contemplated that a test molecule may also modulatetmst2-receptor polypeptide activity indirectly, such as by affectingtmst2 like gene expression, or by binding to an tmst2-receptor ligand.In one embodiment, a test molecule will bind to a tmst2-receptorpolypeptide with an affinity constant of at least about 10⁻⁶M,preferably about 10⁻⁸M, more preferably about 10⁻⁹M, and even morepreferably about 10⁻¹⁰M.

Methods for identifying compounds which interact with tmst2-receptorpolypeptides are encompassed by the invention. In certain embodiments, atmst2-receptor polypeptide is incubated with a test molecule underconditions which permit the interaction of the test molecule totmst2-reccptor polypeptide, and the extent of interaction can bemeasured. The test molecule(s) can be screened in a substantiallypurified form or in a crude mixture.

Test molecules may be nucleic acid molecules, proteins, peptides,carbohydrates, lipids or small molecular weight organic or inorganiccompounds which interacts with tmst2 like polypeptide to regulate itsactivity. Molecules which regulate tmst2-receptor polypeptide expressioninclude nucleic acids which are complementary to nucleic acids encodingan tmst2-receptor polypeptide, or are complementary to nucleic acidssequences which direct or control the expression of tmst2-receptorpolypeptide, and which act as anti-sense regulators of expression. Oncea set of test molecules has been identified as interacting with atmst2-receptor polypeptide, the molecules may be further evaluated fortheir ability to increase or decrease tmst2-receptor activity.

The measurement of the interaction of test molecules with tmst2-receptorpolypeptides may be carried out in several formats, including cell-basedbinding assays, membrane binding assays, solution-phase assays andimmunoassays. In general, test molecules are incubated with atmst2-receptor polypeptide for a specified period of time and the extentof binding to a tmst2-receptor polypeptide is determined by filtration,electrochemiluminescent (ECL, ORIGEN system by IGEN), cell-based orimmunoassays.

Homogenous assay technologies for radioactivity (SPA; Amersham) and timeresolved fluorescence (HTRF, Packard) can also be implemented. Bindingcan be detected by labeling with radioactive isotopes (¹²⁵I,³⁵S, ³H),fluorescent dyes (fluorescein), lanthanides such as Europium (Eu³⁻)chclates or cryptates, orbipyridyl-ruthenium (Ru²⁻) complexes. It isunderstood that the choice of a labeled probe will depend upon thedetection system used. Alternatively, a tmst2-receptor polypeptide maybe modified withi an unlabeled epitope tag (e.g., biotin, peptides,His6, myc, Fc) and bound to proteins such as streptavidin, anti-peptideor anti-protein antibodies which have a detectable label as describedabove.

The interaction of test molecules to tmst2-receptor polypeptides mayalso be assayed directly using polyclonal or monoclonal antibodies in animmunoassay. Alternatively, modified forms of tmst2-receptorpolypeptides containing epitope tags as described herein may be used insolution and immunoassays.

In one embodiment, a tmst2-receptor agonist or antagonist may be aprotein, peptide, carbohydrate, lipid or small molecular weight moleculewhich interacts with tmst2-receptor to regulate its activity. Potentialprotein antagonists of tmst2-receptor include antibodies which bind toactive regions of the polypeptide and inhibit or eliminate at least onceactivity of tmst2-receptor. Molecules which regulate tmst2-receptorpolypeptide expression may include nucleic acids which are complementaryto nucleic acids encoding a tmst2-receptor polypeptide, or arecomplementary to nucleic acids sequences which direct or controlexpression of polypeptide, and which act as anti-sense regulators ofexpression.

In the event that tmst2-receptor polypeptides display biologicalactivity through an interaction with a ligand, a variety of in vitroassays may be used to measure binding of a tmst2-receptor polypeptide tothe corresponding binding partner (such as a selective binding agent orligand). These assays may be used to screen test molecules for theirability to increase or decrease the rate and/or the extent of binding ofa tmst2-receptor polypeptide to its binding partner. In one assay, atmst2-receptor polypeptide is immobilized in the bottom of the wells ofa microtiter plate. Radiolabeled tmst2-receptor binding partner (forexample, iodinated tmst2-receptor binding partner) and the testmolecule(s) can then be added either one at a time (in either order) orsimultaneously to the wells. After incubation, the wells can be washedand counted, using a scintillation counter for, radioactivity todetermine the extent to which the binding partner bound to tmst2receptor polypeptide. Typically, the molecules will be tested over arange of concentrations, and a series of control wells lacking one ormore elements of the test assays can be used for accuracy in theevaluation of the results. An alternative to this method involvesreversing the “positions” of the proteins, i.e., immobilizingtmst2-receptor binding partner to the microtiter plate wells, incubatingwith the test molecule and radiolabeled tmst2-receptor and determiningthe extent of tmst2-receptor binding (see, for example, Chapter 18 ofCurrent Protocols in Molecular Biology, Ausubel et al., eds., John Wiley& Sons, New York, N.Y. (1995)).

As an alternative to radiolabelling, an tmst2-receptor polypeptide orits binding partner may be conjugated to biotin and the presence ofbiotinylated protein can then be detected using streptavidin linked toan enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase(AP), that can be detected colorimetrically, or by fluorescent taggingof streptavidin. An antibody directed to an tmst2-receptor polypeptideor to an tmst2-receptor binding partner and is conjugated to biotin mayalso be used and can be detected after incubation with enzyme-linkedstreptavidin linked to AP or HRP.

A tmst2-receptor polypeptide and a tmst2-receptor binding partner canalso be immobilized by attachment to agarose beads, acrylic beads orother types of such solid phase substrates. The substrate-proteincomplex can be placed in a solution containing the complementary proteinand the test compound; after incubation, the beads can be precipitatedby centriftigation, and the amount of binding between an tmst2-receptorpolypeptide and its binding partner can be assessed using the methodsdescribed herein. Alternatively, the substrate-protein complex can beimmobilized in a column and the test molecule and complementary proteinare passed through the column. The formation of a complex between antmst2-receptor polypeptide and its binding partner can then be assessedusing any of the techniques set forth herein, i.e., radiolabeling,antibody binding, or the like.

Another in vitro assay that is useful for identifying a test moleculewhich increase or decrease the formation of a complex between atmst2-receptor binding protein and a tmst2-receptor binding partner is asurface plasmon resonance detector system such as the Biacore assaysystem (Uppsula. Sweden). The Biacore system may be carried out usingthe manufacturer's protocol. This assay essentially involves thecovalent binding of either tmst2-receptor or a tmst2-receptor bindingpartner to a dextran-coated sensor chip which is located in a detector.The test compound and the other complementary protein can then beinjected either simultaneously or sequentially into the chambercontaining the sensor chip and the amount of complementary protein thatbinds can be assessed based on the change in molecular mass which isphysically associated with the dextran-coated side of the sensor chip;the change in molecular mass can be measured by the detector system.

In some cases, it may be desirable to evaluate two or more testcompounds together for their ability to increase or decrease formationof a complex between a tmst2-receptor polypeptide and a tmst2-receptorbinding partner complex. In these cases, the assays set forth above canbe readily modified by adding such additional test compound(s) eithersimultaneous with, or subsequent to, the first test compound. Theremainder of steps in the assay are as set forth herein.

In vitro assays such as those described above may be used advantageouslyto screen rapidly large numbers of compounds for effects on complexformatioll by tmst2-receptor and tmst2-receptor binding partner. Theassays may be automated to screen compounds generated in phage display,synthetic peptide and chemical synthesis libraries.

Compounds which increase or decrease the formation of a complex betweena tmst2-receptor polypeptide and a tmst2-receptor binding partner mayalso be screened in cell culture using cells and cell lines expressingeither tmst2-receptor or tmst2-receptor binding partner. Cells and celllines may be obtained from any mammal, but preferably will be from humanor other primate, canine, or rodent sources. The binding of antmst2-receptor polypeptidc to cells expressing tmst2-receptor bindingpartner at the surface is evaluated in the presence or absence of testmolecules and the extent of binding may be determined by, for example,flow cytometry using a biotinylated antibody to an tmst2-receptorbinding partner. Cell culture assays may be used advantageously tofurther evaluate compounds that score positive in protein binding assaysdescribed herein.

Cell cultures can also be used to screen the impact of a drug candidate.For example, drug candidates may decrease or increase the expression ofthe tmst2-receptor gene. In certain embodiments, the amount oftmst2-receptor polypeptide that is produced may be measured afterexposure of the cell culture to the drug candidate. In certainembodiments, one may detect the actual impact of the drug candidate onthe cell culture. For example, the overexpression of a particular genemay have a particular impact on the cell culture. In such cases, one maytest a drug candidate's ability to increase or decrease the expressionof the gene or its ability to prevent or inhibit a particular impact onthe cell culture. In other examples, the production of a particularmetabolic product such as a fragment of a polypeptide, may result in, orbe associated with, a disease or pathological condition. In such cases,one may test a drug candidate's ability to decrease the production ofsuch a metabolic product in a cell culture.

A yeast two hybrid system (Chien et al., Proc. Natl. Acad. Sci. USA,88:9578-9583 (1991)) can be used to identify novel polypeptides thatbind to, or interact with, tmst2-receptor polypeptides. As an example,hybrid constructs comprising DNA encoding a cytoplasmic domain of antmst2-receptor polypeptide fused to a yeast GAL4-DNA binding domain maybe used as a two-hybrid bait plasmid. Positive clones emerging from thescreening may be characterized further to identify interacting proteins.

P38 Inhibitors

A new approach to intervention between the extracellular stimulus andthe secretion of IL-I and TNFα from the cell involves blocking signaltransduction through inhibition of a kinase which lies on the signalpathway. One example is through inhibition of P-38 (also called “RK” or“SAPK-2”, Lee et al., Nature, 372:739 (1994)), a known ser/thr kinase(clone reported in Han et al., Biochimica Biophysica Acta, 1265:224-227(1995)). A linear relationship has been shown for effectiveness in acompetitive binding assay to P-38, and the same inhibitor diminishingthe levels of IL-1 secretion from monocytes following LPS stimulation.Following LPS stimulation of monocytes, the levels of messenger RNA forTNF-a have been shown to increase 100 fold, but the protein levels ofTNF-α are increased 10,000 fold. Thus, a considerable amplification ofthe TNF signaling occurs at the translational level. Following LPSstimulation of monocytes in the presence of a P-38 inhibitor, the levelsof mRNA are not affected, but the levels of final TNF protein aredramatically reduced (up to 80-90% depending on the effectiveness of theP-38 inhibitor). Thus, the above experiments lend strong support to theconclusion that inhibition of P-38 leads to diminished translationalefliciency. Further evidence that TNFα is under translational control isfound in the deletion experiments of Beutler et al. and Lee, whereinsegments of 3′ untranslated mRNA (3′ UTR) are removed resulting in hightranslational efficiency for TNFα. More importantly, the P-38 inhibitorsdid not have an effect on the level of TNFα (i.e., translationalefficiency) when the appropriate segments of TNFα mRNA are deleted.Thus, the correlative data between the level of binding of inhibitors toP-38 and the diminished IL-1 and TNFα levels following LPS stimulationwith the same inhibitors, plus the above biochemical evidence regardingthe effect of P-38 inhibitors on translational efficiency of both TNFαand IL-1 make a strong cause and effect relationship. The role of P-38in the celi is still being delineated; so therefore, other beneficialeffects regarding inflammatory diseases or other disease states obtainedfrom its inhibition maybe forthcoming.

Elevated levels of TNFα and/or IL-1 may contribute to the onset,etiology, or exacerbate a number of disease states, including, but notlimited to: rheumatoid arthritis; osteoarthritis; rheumatoidspondylitis; gouty arthritis; inflammatory bowel disease; adultrespiratory distress syndrome (ARDS); psoriasis; Crohn's disease;allergic rhinitis; ulcerative colitis; anaphylaxis; contact dermatitis;asthma; antiviral therapy including those virses sensitive to TNFαinhibition—HIV-1, HIV-2, HIV-3, cytomegalovirus (CMV), influenza,adenovirtlis, and the herpes viruses including HSV-1, HSV-2, and herpeszoster; muscle degeneration; cachexia; Reiter's syndrome; type IIdiabetes; bone resorption diseases; graft vs. host reaction; ischemiareperfusion injury; atherosclerosis; brain trauma; Alzheimer's disease;multiple sclerosis; cerebral malaria; sepsis; septic shock; toxic shocksyndrome; fever and mylagias due to infection.

Substituted imidazole, pyrrole, pyridine, pyrimidine and the likecompounds have been described for use in the treatment of cytokinemediated diseases by inhibition of proinflammatorycytokines, such asIL-1, IL-6, IL-8 and TNF. Substituted imidazoles for use in thetreatment of cytokine mediated diseases have been described in U.S. Pat.No. 5,593,992; WO 93/14081; WO 97/18626; WO 96/21452; WO 96/21654; WO96/40143; WO 97/05878; WO 97/05878; (each of which is incorporatedherein by reference in its entirety). Substituted imidazoles for use inthe treatment of inflammation has been described in U.S. Pat. No.3,929,807 (which is incorporated herein by reference in its entirety).Substituted pyrrole compounds for use in the treatment of cytokinemediated diseases have been described in WO 97/05877; WO 97/05878; WO97/16426; WO 97/16441; and WO 97/16442 (each of which is incorporatedherein by reference in its entirety). Substituted aryl and heteroarylfused pyrrole compounds for use in the treatment of cytokine mediateddiseases have been described in WO 98/22457 (which is incorporatedherein by reference in its entirety). Substituted pyridine, pyrimidine,pyrimidinone and pyridazine compounds for use in the treatment ofcytokine mediated diseases have been described in WO 98/24780; WO98/24782; WO 99/24404; and WO 99/32448 (each of which is incorporatedherein by reference in its entirety).

Internalizing Proteins

The TATprotein sequence (from HIV) can be used to internalize proteinsinto a cell by targeting the lipid bi-layer component of the cellmembrane. See e.g., Falwell et al., Proc. Natl. Acad. Sci., 91: 664-668(1994). For example, an 11 amino acid sequence (YGRKKRRQRRR; SEQ ID NO:15) of the HIV TAT protein (termed the “protein transduction domain”, orTAT PDT) has been shown to mediate delivery of large bioactive proteinssuch as β-galactosidase and p27Kip across the cytoplasmic membrane andthe nuclear membrane of a cell. See Schwarze et al., Science, 285:1569-1572 (1999); and Nagahara et al., Nature Medicine, 4: 1449-1452,(1998). Schwartze et al. (Science, 285: 1569-72 (1999)) demonstratedthat cultured cells acquired β-gal activity when exposed to a fusion ofthe TAT PDT and β-galactosidase. Injection of mice with the TAT-β-galfusion proteins resulted in β-gal expression in a number of tissues,including liver, kidney, lung, heart, and brain tissue.

It will thus be appreciated that the TAT protein sequence may be used tointernalize a desired protein or polypeptide into a cell. In the contextof the present invention, the TAT protein sequence can be fused toanother molecule such as a tmst2-receptor antagonist (i.e.:anti-tmst2-receptor selective binding agent or small molecule) andadministered intracellularly to inhibit the activity of thetmst2-receptor molecule. Where desired, the tmst2-receptor proteinitself, or a peptide fragment or modified form of tmst2-receptor may befused to such a protein transducer for administrating to cells using theprocedures, described herein.

Cell Source Identification Using tmst2-Receptor Polypeptide

In accordance with certain embodiments of the invention, it may beuseful to be able to determine the source of a certain cell typeassociated with an tmst2-receptor polypeptide. For example, it may beuseful to determine the origin of a disease or pathological condition asan aid in selecting an appropriate therapy.

Tmst2-receptor polypeptide is specifically associated with bone marrowassociated stromal cells. Thus, if one knows that particular cellsproduce tmst2-receptor polypeptide or contain nucleic acids that encodetmst2-receptor polypeptide, one will know that such cells are derivedfrom the marrow. According to certain embodiments, it may be useful tobe able to deterniine the source of a certain cell type. For example, itmay be useful to deteniine the origin of a disease or pathologicalcondition which may aid in selecting appropriate therapy. In certainembodiments, nucleic acid encoding tmst2-receptor polypeptide can beused as a probe to identify bone marrow-derived cells by screening thenucleic acids of the cells with such a probe. In other embodiments, onemay use the tmst2-receptor polypeptide to make antibodies that arespecific for tmst2-receptor polypeptide. Such antibodies can be used totest for the presence of tmst2-receptor polypeptide in cells, and thus,used to determine if such cells are marrow derived.

Tmst2-receptor-Polypeptide Compositions and Administration

Members of the TNF ligand family have been implicated in mediation of anumber of diseases. The pleiotropic nature of the TNF and related ligandfamily prevents generalization about whether it is beneficial orinjurious. It is clear that in some instances, the local effects of TNFand other members of the TNF-ligand family cytokines improve hostdefense mechanisms by mobilizing substrate, increasing immune cellfunction, stimulating inflammation and in killing cancer cells. However,in other cases the toxicity of TNF and related cytokines may causedisease by mediating shock, tissue injury, or catabolic injury. Thereare many diseases wherein members of the TNF ligand family mediatedinjury-may be treated or ameliorated by the administration of, solubleforms of the receptor or other ligand binding molecules. These diseasesinclude acquired-immunodeficiency syndrome (AIDS), anemia, autoimmunediseases, cachcxia, cancer, cerebral malaria, diabetes mellitus,disseminated intravascular coagulopathy, erythroid sick syndrome,hemorrhagic shock, hepatitis, insulin resistance, leprosy, leukemia,lymphoma. meningitis, multiple sclerosis, myocardial ischaemia, obesity,rejection of transplanted organs, rheumatoid arthritis, septic shocksyndromiie, stroke, adult respiratory distress syndrome (ARDS),tuberculosis, and a number of viral diseases.

Pharmaceutical compositions of tmst2-reccptor polypeptides are withinthe scope of the present invention for prophylactic and therapeutictreatment of humans and animals for indications resulting from abnormalexpression of tmst2-receptor or where it is determined thatadministration of tmst2-receptor polypeptide will result in theamelioration or cure of the indications. Such tmst2-receptorpharmaceutical compositions may comprise a therapeutically effectiveamount of a tmst2-receptor polypeptide and/or its binding partner, ortherapeutically active fragment(s), variant(s), or derivative(s) thereofin admixture with a pharmaceutically or physically acceptable additivesand/cr carriers. Suitable formulation materials or pharmaceuticallyacceptable agents include, but are not limited to, antioxidants,preservatives, colors, flavoring, and diluting agents, emulsifyingagents, suspending agents. solvents. fillers, bulking agents, buffers,deliverv vehicles, diluents, excipients, and/or pharmaceuticaladjuvants. Typically, a therapeutic compound containing tmst2-receptorpolypeptide(s) will be administered in the form of a compositioncomprising purified polypeptide, fragment(s), variant(s), orderivative(s) in conjunction with one or more physiologically acceptablecarriers, excipients, or diluents. For example, a suitable vehicle maybe water for injection, physiological solution, or artificialcerebrospinal fluid possibly supplemented with other materials common incompositions for parenteral delivery.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. Preferably, the product is formulated as alyophilizate usinty appropriate excipients (e.g., sucrose). Otherstandard carriers, diluents, and excipients may be included as desired.Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5,or acetate buffer of about pH 4.0-5.5, which may further includesorbitol or a suitable substitute therefor. The pH of the solutionshould also be selected based on the relative solubility of tmst2 atvarious pHs.

The primary solvent in a composition may be either aqueous ornon-aqueous in nature. In addition, the vehicle may contain otherformulation materials for modifying or maintaining the pH, osmolarity,viscosity, clarity, color, isotonicity, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the composition maycontain additional formulation materials for modifying or maintainingthe rate of release of tmst2-receptor protein, or for promoting theabsorption or penetration of tmst2-receptor protein.

Compositions comprising the tmst2-receptor polypeptide compositions canbe administered parentally. Alternatively, the compositions may beadministered intravenously or subcutaneously. When systemicallyadministered, the therapeutic compositions for use in this invention maybe in the form of a pyrogen-free, parentally acceptable aqueoussolution. The preparation of such phaniaceutically acceptable proteinsolutions, with due regard to pH, isotonicity, stability and the like,is within the skill of the art.

Therapeutic formulations of tmst2-receptor polypeptide compositionsuseful for practicing the present invention may be prepared for storageby mixing the selected composition having the desired degree of puritywith optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 18th Edition, A. R.Gennaro, ed., Mack Publishing Company [1990]) in the fom of alyophilized cake or an aqueous solution.

Acceptable carriers, excipients or stabilizers are nontoxic torecipients and are preferably inert at the dosages and concentrationsemployed, and include buffers (such as borate, bicarbonate, Trsi-HCl,phosphates, citrates, or other organic acids); antioxidants (such asascorbic acid, sodium sulfite or hydrogen sulfite); low molecular weightpolypeptides; proteins (such as serum albumin, gelatin, orimmunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone);amino acids (such as glycine, glutamine, asparagine, arginine orlysine); fillers; monosaccharides, disaccharides, and othercarbohydrates (such as glucose, mannose, or dextrins); chelating agents(such as EDTA); sugar alcohols (such as mannitol or sorbitol);salt-forming counterions (such as sodium); and/or nonionic surfactants(such as Tween, plur-onics or polyethylene glycol (PEG)).

An effective amount of the tmst2-receptor polypeptide(s) composition tobe employed therapeutically will depend, for example, upon thetherapeutic objectives such as the indication for which the compositionis being used, the route of administration (e.g., whether it isadministered locally or systemically), and the condition ol the patient(e.g., patient's general health, anaureuesis, age, weight, sex). It Isessential, when detenninilln the therapeutically effective dose, to takeinto account the quantity of tmst2-receptor or other members of the TNFfamily of ligand secreted which are responsible for the disease as wellas the quantity of endogenous tmst2-receptor. Basically, it can beassumed that for effective treatment of a disease triggered by thesecretion of thei cytokine(s), at least the same molar amount of thetmst2-receptor polypeptide(s) is required as quantity of ligandsecreted, and possibly a multiple excess might be needed, although lessmay be needed depending on the nature of the specific ligand involvedand the nature of its interaction with tmst2-receptor. Accordingly, itwill be necessary for the therapist to titer the dosage and/or in vivimodify the route of administration as required to obtain the optimaltherapeutic effect. A typical daily dosage may range from about 0.1mg/kg to up to 100 mg/kg or more. depending on the factors mentionedabove. Typically, a clinician will administer the composition until adosage is reached that achieves the desired effect. The composition maytherefore be administered as a single dose, or as two or more doses(which may or may not contain the same amount of tmst2-receptorpolypeptide) over time, or as a continuous infusion via implantationdevice or catheter.

An effective amount of an tmst2-receptor pharmaceutical composition tobe employed therapeutically will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the tmst2-receptor molecule is being used, the route ofadministration, and the size (body weight, body surface or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician may titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect.

The tmst2-receptor polypeptide composition to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes. Where the compositionis lyophilized, sterilization using these methods may be conductedeither prior to or following lyophilization and reconstitution. Thecomposition for parenteral administration ordinarily will be stored inlyophilized form or in solution.

Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

Once the phanmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such fonrulations may be storedeither in a ready-to-usc form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

Effective administration forms, such as (I) slow-release fonnulations,(2) inhalant mists, or (3) orally active formulations are alsoenvisioned. Pharmaceutical composition comprising therapeuticallyeffective dose of the tmst2-receptor polypeptide also may be formulatedfor parenteral administration. Such parenterally administeredtherapeutic compositions are typically in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising tmst2-receptor in apharmaceutically acceptable vehicle. The tmst2-receptor pharmaceuticalcompositions also may include particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, etc. or theintroduction of tmst2-reccptor into liposomes. Hyaluronic acid may alsobe used, and this may have the effect of promoting sustained duration inthe circulation.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired tmst2-receptor molecule in a pharmaceutically acceptablevehicle. A particularly suitable vehicle for parenteral injection issterile distilled water in which an tmst2-receptor molecule isformulated as a sterile, isotonic solution, properly preserved. Yetanother preparation can involve the formulation of the desired moleculewith an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (polylactic acid,polyglycolic acid), or beads, orliposomes, that provides for the controlled or sustained release of theproduct which may then be delivered as a depot injectioni. Ilyaluronicacid may also be used, and this may have the effect of promotingsustained duration in the circulation. Other suitable means for theintroduction of the desired molecule include implantable drug deliverydevices.

The preparations of the present invention may include other components,for example parenterally acceptable preservatives, tonicity agents,cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, antioxidants and surfactants, as are well known in theart. For example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol and the like. Suitable preservatives include, but arenotlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsare for example glycerin, propylene glycol and polyethylene glycol.Suitable complexing agents are for example caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamnine, lecithin, cholesterol, tyloxapal and the like. Thebuffers can be conventional buffers such as borate, citrate, phosphate,bicarbonate, or Tris-HCl.

The formulation components are present in concentration that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at slightly lower pH,typically within a pH range of from about 5 to about 8.

In one embodiment, a pharmaceutical composition may be fonnulated forinhalation. For example, tmst2-receptor may be formulated as a drypowder for inhalation. Tmst2-receptor polypeptide or tmst2-receptorpolynucleotide inhalation solutions may also be formulated with apropellant for aerosol delivery. In yet another embodiment, solutionsmay be nebulized. Pulmonary administration is further disclosed in PCTapplication No. PCT/US94/001875 which discusses pulmonary deleivery ofchemically modified proteins.

It is also contemplated that certain formulations containingtmst2-receptor can be administered orally. The tmst2-receptor which isadministered in this fashion may be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degrad(ation is minimized. Additional agents may beincluded to facilitate absorption of the receptor polypeptide. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed.

Another phanmaceutical composition may involve an effective quantity oftmst2-receptor in a mixture with non-toxic excipicnts which are suitablefor the manufacture of tablets. By dissolving the tablets in sterilewater, or other appropriate vehicle, solutions can be prepared in unitdose form. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional tmst2-receptor phanracCutical compositions will be evident tothose skilled in the art, including formulations involvingtmst2-receptor in sustained- or controlled-release deliveryfonmulations. Techniques for formulating a variety of other sustained-or controlled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, PCT/US93/00829 whichdiscloses controlled release of porous polymeric microparticles for thedelivery of pharmaceutical compositions. Additional examples ofsustained-release preparations include semipermeable polymer materialsin the form of matrices shaped articles e.g., film or microcapsules.

Regardless of the manner of administration, the specific dose may becalculated according to body weight, body surface area or organ size.Further refinement of the calculations necessary to determinc theappropriate dosage for treatment involving each of the above mentionedfonmulations is routinely made by those of ordinary skill in the art andis within the ambit of tasks routinely performed by them. Appropriatedosages may be ascertained through use of appropriate dose-responsedata. A typical dosage may range from about 0.1 mg/kg to up to about 100mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 0.1 mg/kg up to about 100 mg/kg;or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100 mg/kg.

The frequency ofdosin(g will depend upon the pharmacokitnetic parametersof the tmst2-receptor molecule in the fonnulation used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

The route of administration of the composition is in accord with knownmethods, e.g. oral, injection or infusion by intravenous,intraperitoneal, intracerebral (intraparenchymal), intraventricular,intramuscular, intraocular, intraarterial, or intralesional routes, orby sustained release systems or implantation device which may optionallyinvolve the use of a catheter. Where desired, the compositions may beadministered continuously by infusion, bolus injection or byimplantation device. Alternatively or additionally, the composition maybe administered locally via implantation into the affected area of amembrane, sponge, or other appropriate material on to whichtmst2-receptor polypeptide has been absorbed.

One may further administer the present pharmaceutical compositions bypulmonary administration, see, e.g., International Publication No: WO94/20069, which discloses pulmonary delivery of chemically modifiedproteins, herein incorporated by reference. For ptilmonary delivery, theparticle size should be suitable for delivery to the distal lung. Forexample, the particle size may be from 1 mm to 5 mm, however, largerparticles may be used, for example, if each particle is fairly porous.Alternatively or additionally, the composition may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which receptor polypeptide has beenabsorbed or encapsulated. Where an implantation device is used, thedevice may be implanted into any suitable tissue or organ, and deliverymay be directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

Tmst2-receptor polypeptide and/or its binding partner may also beadministered In a sustained release formulation or preparation. Suitablepolymer compositions preferably have intrinsic and controllablebiodegradability so that they persist for about a week to about sixmonths; are non-toxic containing no significant toxic monomers anddegrading into non-toxic components; are biocompatible, are chemicallycompatible with substances to be delivered, and tend not to denature theactive substance; are sufficiently porous to allow the incorporation ofbiologically active molecules and their subsequent liberation from thepolymer by diffusion, erosion or a combination thereof; are able toremain at the site of the application by adherence or by geometricfactions, such as being fonmed in place or softened and subsequentlymolded or fonmed into microparticles which are trapped at a desiredlocation; are capable of being delivered by techniques of minimuminvasivity such as by catheter, laparoscope or endoscope. Sustainedrelease matrices include polyesters, hydrogels, polylactides (U.S. Pat.No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al, Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid.(EP 133,988). Sustained-release compositions also may include liposomes,which can be prepared by any of several methods known in the art (e.g.,Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985)1; EP36,676; EP 88,046; EP 143,949, incorporated herein by reference).

The tmst2-receptor polypeptides, variants, derivatives or fragmentsthereof, may be employed alone, together, or in combination with otherphannaceutical compositions. The tmst2-receptor polypeptides, fragments,variants, and derivatives may be used in combination with cytokines,cytokine inhibitors, growth factors, antibiotics, anti-inflammatories,and/or chemotherapeutic agents as is appropriate for the indicationbeing treated

In some cases, it may be desirable to use tmst2-receptor polypeptidecompositions in an ex vivo manner. Here, cells, tissues, or organs thathave been removed from the patient are exposed to tmst2-receptorpolypeptide compositions after which the cells, tissues and/or organsare subsequently implanted back into the patient.

In other cases, a tmst2-receptor polypeptide can be delivered byimplalltinlo into patients certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptides, fragments, variants, or derivatives. Suchcells may be animal or human cells, and may be autologous. heretologous,or xenogeneic. Optionally, the cells may be immortalized. In order todecrease the chance of an immunological response, it is preferred thatthe cells be encapsulated to avoid infiltration of surrounding tissues.The encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

Methods used for membrane encapsulation of cells are familiar to theskilled artisan, and preparation of encapsulated cells and theirimplantation in patients may be accomplished without undueexperimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and5,106,627, incorporated herein by reference. A system for encapsulatingliving cells is described in International Publication No: WO 91/10425(Aebischer et al.). Techniques for formulating a variety of othersustained or controlled delivery means, such as liposome carriers,bio-erodible particles or beads, are also known to those in the art, andare described, for example, in U.S. Pat. No. 5,653,975, incorporatedherein by reference. The cells, with or without encapsulation, may beimplanted into suitable body tissues or organs of the patient.

As discussed above, it may be desirable to treat isolated cellpopulations such as stem cells, lymphocytes, red blood cells,chondrocytes, neurons, and the like; add as appropriate with one or moretmst2-receptor polypeptides, variants, derivatives and/or fragments.This can be accomplished by exposing the isolated cells to thepolypeptide, variant, derivative, or fragment directly, where it is in aform that is perrneable to the cell membrane.

The present invention relates to improved methods for both the in vitroproduction of therapeutic proteins and for the production and deliveryof therapeutic proteins by gene therapy.

Additional embodiments ofthe present invention relate to cells andmethods (e.g., homologous recombination and/or other recombinantproduction methods) for both the in vitro production of therapeuticpolypeptides and for the production and delivery of therapeuticpolypeptides by gene therapy or cell therapy. Homologous and otherrecombination methods may be used to modify a cell that contains anormally transcriptionally silent tmst2-reccptor gyene, or an underexpressed gene, and thereby produce a cell which expressestherapeutically efficacious amounts of tmst2-receptor polypeptides.

Homologous Recombination

It is further envisioned that tmst2-receptor protein may be produced byhomologous recombination, or with recombinant production methodsutilizing control elements introduced into cells already containing DNAencoding tmst2-receptor. For example, homologous recombination methodsmay be used to modify a cell that contains a normally transcriptionallysilent tmst2-receptor gene, or an under expressed gene, and therebyproduce a cell which expresses therapeutically efficacious amounts oftmst2-receptor. Homologous recombination is a technique originallydeveloped for targeting genes to induce or correct mutations intranscriptionally active genes (Kucherlapati, Prog. in Nucl. Acid Res.and Mol. Biol., 36:301, (1989)). The basic technique was developed as amethod for introducing specific mutations into specific regions of themammalian genoine (Thomas et al., Cell, 44:419-428, (1986); Thomas andCapecchi, Cell, 51:503-512, (1987); Doetschman et al., Proc. Natl. Acad.Sci U.S.A., 85:8583-8587, (1988)) or to correct specific mutationswithin defective genes (Doetschman et al., Nature, 330:576-578, (1987)).Exemplary homologous recombination techniques are described in U.S. Pat.No. 5,272,071, EP Publication No: 9193051, EP Publication No. 505 500;PCT/US90/07642, International Publication No: WO 91/09955, incorporatedherein by reference.

Through homologous recombination, the DNA sequence to be inserted intothe genome can be directed to a specific region of the gene of interestby attaching it to targeting DNA. The targeting DNA is a nucleotidesequence that is complementary (homologous) to a region of the genomicDNA. Small pieces of targeting DNA that are complementary to a specificregion of the genome are put in contact with the parental strand duringthe DNA replication process. It is a general property of DNA that hasbeen inserted into a cell to hybridize, and therefore, recombine withother pieces of endogenous DNA throLugh shared homologous regions. Ifthis complementary strand is attached to an oligonucleotide thatcontains a mutation or a different sequence or an additional nucleotide,it too is incorporated into the newly synthesized strand as a result ofthe recombination. As a result of the proofreading function, it ispossible for the new sequence of DNA to serve as the template. Thus, thetransferred DNA is incorporated into the genome.

Attached to these pieces of targeting DNA are regions of DNA which mayinteract with or control the expression of a tmst2-receptor polypeptide,e.g., flanking sequences. For example, a promoter/enhancer element, asuppresser, or an exogenous transcription modulatory element is insertedin the genome of the intended host cell in proximity and orientationsufficient to influence the transcription of DNA encoding the desiredtmst2-receptor polypeptide. The control element controls a portion ofthe DNA present in the host cell genome. Thus, the expression oftmst2-receptor polyepetide may be achieved not by transfection of DNAthat encodes the tmst2-receptor gene itself, but rather by the usc oftargeting DNA (containing regions of homology with the endogenous geneof interest) coupled with DNA regulatory segments that provide theendogenous gene sequence with recognizable signals for transcription ofa tmst2-receptor protein.

In an exemplary method, expression of a desired targeted gene in a cell(i.e., a desired endogenous cellular gene) is altered via homologousrecombination into the cellular genome at a preselected site, by theintroduction of DNA which includes at least a regulatory sequence, anexon and a splice donor site. These components are introduced into thechromosomal (genomic) DNA in such a manner that this, in effect, resultsin the production of a new transcription unit (in which the regulatorysequence, the exon and the splice donor site present in the DNAconstruct are operatively linked to the endogenous gene). As a result ofthe introduction of these components into the chromosomal DNA, theexpression of the desired endogenous gene is altered.

Altered gene expression, as described herein, encompasses activating (orcausing to be expressed) a gene which is noirnally silent (unexpressed)in the cell as obtained, as well as increasing expression of a genewhich is not expressed at physiologically significant levels in the cellas obtained. The embodiments further encompass changing the pattern ofregulation or induction Such that it is different from the pattern ofregulation or induction that occurs in the cell as obtained, andreducing (including eliminating) expression of a gene which is expressedin the cell as obtained.

One method by which homologous recombination can be used to increase, orcause, tmst2-receptor polypeptide production from a cell's endogenoustmst2-receptor gene involves first using homologous recombination toplace a recombination sequence from a site-specific recombination system(e.g., Cre/loxP, FLP/FRT) (Sauer, Curr. Opin. Biotech., 5:521-527,(1994); Sauer, Meth. Enz., 225:890-900, (1993)) upstream (that is, 5′to) of the cell's endogenous genomic tmst2-receptor polypeptide codingregion. A plasmid containing a recombination site homologous to the sitethat was placed just upstream of the genomic tmst2-receptor polypeptidecoding region is introduced into the modified cell line along with theappropriate recombinase enzyme. This recombinase causes the plasmid tointegrate, via the plasmid's recombination site, into the recombinationsite located just upstream of the genomic tmst2-receptor polypeptidecoding region in the cell line (Baubonis and Saner, Nucleic Acids Res.,21:2025-2029, (1993); O'Gorman et al., Science, 251:1351-1355, (1991)).Any flanking sequences known to increase transcription (e.g.,enhancer/promoter, intron, translational enhancer), if properlypositioned in this plasmid, would integrate in such a manner as tocreate a new or modified transcriptional unit resulting in de novo orincreased tmst2-receptor polypeptide production from the cell'sendogenous tmst2-receptor gene.

A further method to use the cell line in which the site specificrecombination sequence had been placed just upstream of the cell'sendogenous genomic tmst2-receptor polypeptide coding region is to usehomologous recombination to introduce a second recombination siteelsewhere in the cell line's genome. The appropriate recombinase enzymeis then introduced into the two-recombination-site cell line, causing arecombination event (deletion, inversion, translocation) (Sauer, Curr.Opin. Biotech., sluprci, 1994; Saner, Meth. Enz., supra, 1993) thatwould create a new or modified transcriptional unit resulting in de novoor increased tmst2-receptor polypeptide production from the cell'sendogenous tmst2-receptor gene.

An additional approach for increasing, or causing, the expression oftmst2-receptor polypeptide from a cell's endogenous tmst2-receptor geneinvolves increasing, or causing, the expression of a gene or genes(e.g., transcription factors) and/or decreasing the expression of a geneor genes (e.g., transcriptional repressors) in a mariner which resultsin de novo or increased tmst2-receptor polypeptide production from thecell's endogenous tmst2-receptor gene. This method includes theintroduction of a non-naturally occulTing polypeptide (e.g., apolypeptide comprising a site specific DNA binding domain fused to atranscriptional factor domain) into the cell such that de novo orincreased tmst2 like polypeptide production from the cell's endogenoustmst2 like gene results.

The present invention further relates to DNA constructs useful in themethod of altering expression of a target gene. In certain embodiments,the exemplary DNA constructs comprise: (a) one or more targetingsequences; (b) a regulatory sequence; (c) an exon; and (d) an unpairedsplice-donor site. The targeting sequence in the DNA construct directsthe integration of elements (a)-(d) into a target gene in a cell suchthat the elements (b)-(d) are operatively linked to sequences of theendogenous target gene. In another embodiment, the DNA constructscomprise: (a) one or more targeting sequences, (b) a regulatorysequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) asplice-acceptor site, wherein the targeting sequence directs theintegration of elements (a)-(f) such that the elements of (b)-(f) areoperatively linked to the endogenous gene. The targeting sequence ishomologous to the preselected site in the cellular chromosomal DNA withwhich homologous recombination is to occur. In the construct, the exonis generally 3′ of the regulatory sequence and the splice-donor site is3′ of the exon. If the sequence of a particular gene is known, such asthe nucleic acid sequence of tmst2-receptor presented herein, a piece ofDNA that is complementary to a selected region of the gene can besynthesized or otherwise obtained, such as by appropriate restriction ofthe native DNA at specific recognition sites bounding the region ofinterest. This piece serves as a targeting sequence upon insertion intothe cell and will hybridize to its homologous region within the genome.If this hybridization occurs during DNA replication, this piece of DNA,and any additional sequence attached thereto, will act as an Okazakifragment and will be backstitched into the newly synthesized daughterstrand of DNA. The present invention, therefore, includes nuclCeotidesencoding a tmst2-receptor molecule, which nucleotides may be used astargeting sequences.

Tmst2-receptor Cell Therapy and Geiie Therapy

Tmst2-receptor cell therapy. e.g., the implantation of cells producingtmst2-receptor, is also contemplated by the present invention. Thisembodiment would involves implanting cells capable of synthesizing andsecreting a biologically active form of the soluble tmst2-receptor. Suchsoluble tmst2-receptor-producing cells can be cells that are naturalproducers of tmst2-receptor polypeptides or may be recombinant cellswhose ability to produce tmst2-receptor has been augmented bytransformation with a gene encoding the desired tmst2-receptor moleculeor with a gene augmenting the expression of tmst2-receptor. Such amodification may be accomplished by means of a vector suitable fordelivering the gene as well as promoting its expression and secretion.In order to minimize a potential immunological reaction in patientsbeing administered a tmst2-receptor polypeptide, as may occur with theadministration of a polypeptide of a foreign species, it is preferredthat the natural cells producing tmst2-receptor be of human origin andproduce human tmst2-receptor polypeptides. Likewise, it is preferredthat the recombinant cells producing tmst2-receptor polypeptidesbetransformed with an expression vector containing a gene encoding a humantmst2-receptor polyepeptides.

Implanted cells may be encapsulated to avoid infiltration of surroundingtissue. Human or non-human animal cells may be implanted in patients inbiocompatible, semipermeable polymeric enclosures or membranes thatallow release of tmst2-receptor but that prevent destruction of thecells by the patient's immune system or by other detrimental factorsfrom the surrounding tissue. Alternatively, the patient's own cells,transformed to produce tmst2-receptor ex vivo, may be implanted directlyinto the patient without such encapsulation.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be routinely accomplished. For example, Baetge et al.PCT/US94/09299 describes membrane capsules containing geneticallyengineered cells for the effective delivery of biologically activemolecules. The capsules encapsulate cell transfected with recombinantDNA molecules comprising DNA sequences coding for biologically activemolecules operatively linked to promoters that are not subject to downregulation in vivo upon implantation into a mammalian host. The capsulesare biocompatible and are easily retrievable. The devices provide forthe delivery of the molecules from living cells to specific sites withina recipient. See U.S. Pat. Nos. 4,892,538, 5,011,472, and 5,106,627,incorporated herein by reference. A system for encapsulating livingcells is described in Aebischer et (it. (WO 91/1042, WO 91/10470); Winnet al., Exper. Neurol., 113:322-329, 1991, Aebisclher et al., Exper.Neurol., 111:269-275, 1991; and Tresco et al., ASAIO, 38:17-23, 1992.

In vivo and in vitro gene therapy delivery of tmst2-receptor is alsoencompassed by the present invention. In vivo gene therapy may beaccomplished by introducing the gene encoding tmst2-receptor into cellsvia local injection of a polyucleotide molecule or other appropriatedelivery vectors. (Hefti, J. Neurobiology,. 25:1418-1435, 1994). Forexample, a polynucleotide molecule encoding tmst2-receptor may becontained in an adeno-associated virus vector for delivery to thetargeted cells (e.g., Johnson, International Publication No. WO95/34670; International Application No. PCT/US95/07178). The recombinantadeno-associated virus (AAV) genome typically contains AAV invertedterminal repeats flanking a DNA sequence encoding tmst2-receptoroperably linked to functional promoter and polyadenylation sequences.

Alternative viral vectors include, but are not limited to, retrovirus,adenovirus, herpes simplex virus and papilloma virus vectors. U.S. Pat.No. 5,672,344 (issued Sep. 30, 1997, Kelley et al., University ofMichigan) describes an in vivo viral-mediated gene transfer systeminvolving a recombinant neurotrophic HSV-1 vector. U.S. Pat. No.5,399,346 (issued Mar. 21, 1995, Anderson et al., Department of Healthand Human Services) provides examples of a process for providing apatient with a therapeutic protein by the delivery of human cells whichhave been treated in vitro to insert a DNA segment encoding atherapeutic protein. Additional methods and materials for the practiceof gene therapy techniques are described in U.S. Pat. No. 5,631,236(issued May 20, 1997, Woo et al., Baylor College of Medicine) involvingadenoviral vectors; U.S. Pat. No. 5,672,510 (issued Sep. 30, 1997,Eglitis et al., Genetic Therapy, Inc.) involving retroviral vectors; andU.S. Pat. No. 5,635,399 (issued Jun. 3, 1997, Kriegler et al., ChironCorporation) involving retroviral vectors expressing cytokines.

Nonviral delivery methods include, but are not limited to,liposome-mediated transfer, naked DNA delivery (direct injection),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation and microparticle bombardment (e.g, genegun). Gene therapy materials and methods may also include induciblepromoters, tissue-specific enhancer-promoters. DNA sequences designedfor site-specific integration, DNA sequences capable of providing aselective advantage over the parent cell, labels to identifytransfonined cells, negative selection systems and expression controlsystems (safety measures), cell-specitic binding agents (for celltargetsin), cell-specific internalization factors, transcription factorsto enhance expression by a vector as well as methods of vectormanufacture. Such additional methods and materials for the practice ofgene therapy techniques are described in U.S. Pat. No. 4,970,154involving electroporation techniques; WO 9640958 involving, nuclearligands; U.S. Pat. No. 5,679,559 describing a lipoprotein-contianingsystem for gene deleivery; U.S. Pat. No. 5,676,954 involving liposomecarriers; U.S. Pat. No. 5,593,875 concerning methods for calciumphosphate transfection; and U.S. Pat. No. 4,945,050 wherein biologicallyactive particles are propelled at cells at a speed whereby the particlespenetrate the surface of the cells and become incorporated into theinterior of the cells.

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the tmst2-receptor gene in the target cell.Such elements are turned on in response to an appropriate effector. Inthis way, a therapeutic polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs (as described in W09641865 (PCT/US96/099486); WO9731898(PCT/US97/03137) and WO9731899 (PCT/US95/03157)) used to dimerizechimeric proteins which contain a small molecule-binding domain and adomain capable of initiating biological process, such as a DNA-bindingprotein or transcriptional activation protein. The dimerization of theproteins can be used to initiate transcription of the transgene.

An alternative regulation technology uses a method of storing proteinsexpressed from the gene of interest inside the cell as an aggregate orcluster. The gene of interest is expressed as a fusion protein thatincludes a conditional aggregation domain which results in the retentionof the aggregated protein in the endoplasmic reticulum. The storedproteins are stable and inactive inside the cell. The proteins can bereleased, however, by administering a drug (e.g., small molecule ligand)that removes the conditional aggregation domain and thereby specificallybreaks apart the aggregates or clusters so that the proteins may besecreted from the cell. See, Science, 287:816-817, and 826-830 (2000).

Other suitable control means or gene switches include, but are notlimited to, the following systems. Mifepristone (RU486) is used asaprogesterone antagonist. Thie binding of a modified progesteronereceptor ligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors which thenpass into the nucleus to bind DNA. The ligand binding domain is modifiedto eliminate the ability of the receptor to bind to the natural ligand.The modified steroid hormone receptor system is further described inU.S. Pat. No. 5,364,791; WO9640911, and WO9710337.

Yet another control system uses ecdysone (a fniit fly steroid hormone)which binds to and activates an ecdysone receptor (cytoplasmicreceptor). The receptor then translocates to the nucleus to bind aspecific DNA response element (promoter from ecdysone-responsive gene).The ecdysone receptor includes a transactivation domain/DNA-bindingdomain/ligand-binding domain to initiate transcription. The ecdysonesystem is further described in U.S. Pat. No. 5,514,578; WO9738117;WO9637609; and WO9303162.

Another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758; 5,650,298 and 5,654,168.

It is also contemplated that tmst2-receptor gene therapy or cell therapycan further include the delivery of a second protein. For example, thehost cell may be modified to express and release soluble forms of bothtmst2-receptor and TNF-α, or tmst2-receptor and IL-1R. Alternatively,the tmst2-receptor and TNF-α, or tmst2-receptor and IL-1R, may beexpressed in and released from separate cells. Such cells may beseparately introduced into the patient or the cells may be contained ina single implantable device, such as the encapsulating membranedescribed above.

One manner in which g,ene therapy can be applied is to use thetmst2-receptor gene (either genomic DNA, cDNA, and/or synthetic DNAencoding a tmst2-receptor polypeptide, or a fragment, variant, orderivative thereof) which may be operably linked to a constitutive orinducible promoter to form a “gene therapy DNA construct”. The promotermay be homologous or heterologous to the endogenous tmst2-receptor gene,provided that it is active in the cell or tissue type into which theconstruct will be inserted. Other components of the gene therapy DNAconstruct may optionally include, as required, DNA molecules designedfor site-specific integration (e.g., endogenous flanking sequencesuseful for homologous recombination), tissue-specific promoter,enhancer(s) or silencer(s), DNA molecules capable of providing aselective advantage over the parent cell, DNA molecules useful as labelsto identify transformed cells, negative selection systems, cell specificbinding agents (as, for example, for cell targeting) cell-specificinternalization factors, and transcription factors to enhance expressionby a vector as well as factors to enable vector manufacture.

This gene therapy DNA construct can then be introduced into thepatient's cells (either er vivo or in vivo). One means for introducingthe gene therapy DNA construct is via viral vectors. Suitable viralvectors typically used in gene therapy for delivery of gene therapy DNAconstructs include, without limitation, adenovirus, adeno-associatedvirus, herpes simplex virus, lentivirus, papilloma virus, and retrovirusvectors. Some of these vectors, such as retroviral vectors, will deliverthe gene therapy DNA construct to the chromosomal DNA of the patient'scells, and the gene therapy DNA construct can integrate into thechromosomal DNA; other vectors will function as episomes and the genetherapy DNA construct will remain in the cytoplasm. The use of genetherapy vectors is described, for example, in U.S. Pat. Nos. 5,672,344;5,399,346; 5,631,236; and 5,635,399, incorporated herein by reference.

Alternative means to deliver gene therapy DNA constructs to a patient'scells without the use of viral vectors include, without limitation,liposome-mediated transfer, direct injection of naked DNA,receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation, and microparticle bombardment (e.g.,“gene gun”). See U.S. Pat. No. 4,970,154; International Application No.WO 96/40958; U.S. Pat. No. 5,679,559; U.S. Pat. No. 5,676,954; and U.S.Pat. No. 5,593,875, incorporated herein by reference.

Another means to increase endogenous tmst2-receptor polypeptideexpression in a ccil via gene therapy is to insert one or more enhancerelements into the tmst2-receptor polypeptide promoter, where theenhancer clement(s) can serve to increase transcriptional activity ofthe tmst2-receptor polypeptides gene. The enhancer element(s) used willbe selected based on the tissue in which one desires to activate thegene(s); enhancer elements known to confer promoter activation in thattissue will be selected. For example, if a tmst2-receptor polypeptide isto be “turned on” in T-cells, the lck promoter enhancer element may beused. Here, the functional portion of the transcriptional element to beadded may be inserted into a fragment of DNA containing thetmst2-receptor polypeptide promoter (and optionally vector, 5′ and/or 3′flanking sequence, etc.) using standard cloning techniques. Thisconstruct, known as a “homologous recombination construct”, can then beintroduced into the desired cells either ex vivo or in vivo.

Gene therapy also can be used to decrease tmst2-receptor polypeptideexpression where desired by modifying the nucleotide sequence of theendogenous promoter(s). Such modification is typically accomplished viahomologous recombination methods. For example, a DNA molecule containingall or a portion of the promoter of the tmst2-receptor gene(s) selectedfor inactivation can be engineered to remove and/or replace pieces ofthe promoter that regulate transcription. For example, the TATA boxand/or the binding site of a transcriptional activator of the promotermay be deleted using standard molecular biology techniques; suchdeletion can inhibit promoter activity thereby repressing transcriptionof the corresponding tmst2-receptor gene. The deletion of the TATA boxor transcription activator binding site in the promoter may beaccomplished by generating a DNA construct comprising all or therelevant portion of the tmst2-receptor polypeptide promoter(s) (from thesame or a related species as the tmst2-receptor gene(s) to be regulated)in which one or more of the TATA box and/or transcriptional activatorbinding site nucleotides are mutated via substitution, deletion and/orinsertion of one or more nucleotides. As a result, the TATA box and/oractivator binding site has decreased activity or is rendered completelyinactive. This construct, which also will typically contain at leastabout 500 bases of DNA that correspond to the native (endogenous) 5′ and3′ DNA sequences adjacent to the promoter seyncnt that has beenmodified. The construct may be introduced into the appropriate cells(either ex vivo or in vivo) either directly or via a viral vector asdescribed herein. Typically, the integration of the construct into thegenornic DNA of the cells will be via homologous recoinbination, wherethe 5′ and 3′ DNA sequences in the promoter construct can serve to helpintegrate the modified promoter region via hybridization to theendogenotis chromosomal DNA.

Other gene therapy methods may also be employed where it is desirable toinhibit the activity of one or more tmst2-receptor polypeptides. Forexample, antisense DNA or RNA molecules, whlilch hanvC a sequence thatis complementary to at least a portion of the selected tmst2-reccptorpolypeptide gene(s) can be introduced into the cell. Typically, eachsuch antisense molecule will be complementary to the start site (5′ end)of each selected tmst2-receptor gene. When the antisense molecule thenhybridizes to the corresponding tmst2-receptor mRNA, translation of thismRNA is prevented or reduced. Antisence inhibitors provide infomiationrelating to the decrease or absence of tmst2-receptor polypeptides in acell or organism.

Alternatively, gene therapy may be employed to create adominant-negative inhibitor of one or more tmst2-receptor polypeptides.In this situation, the DNA encoding a mutant full length or truncatedpolypeptide of each selected tmst2-receptor polypeptide can be preparedand introduced into the cells of a patient using either viral ornon-viral methods as described herein. Each such mutant is typicallydesigned to compete with endogenous polypeptide in its biological role.

In addition, an tmst2-receptor polypeptidc, whether biologically activeor not, may be used as an immunogen, that is, the polypeptide containsat least one epitope to which antibodies may be raised. Selectivebinding agents that bind to an tmst2-receptor polypeptide (as describedherein) may be used for in vivo and in vitro diagnostic purposes,including, but not limited to, use in labeled form to detect thepresence of tmst2-receptor polypeptide in a body fluid or cell sample.The antibodies may also be used to prevent, treat, or diagnose a numberof diseases and disorders, including those recited herein. Theantibodies may bind to an tmst2-receptor polypeptide so as to diminishor block at least one activity characteristic of an tmst2 likepolypeptide, or may bind to a polypeptide to increase at least oneactivity characteristic of an tmst2-i-eceptor polypeptide (including byincreasing the pharmnacokinetics of the tmst2-receptor polypeptide).

Additional Uses of tmst2 like Nucleic Acids and Polypeptides

Nucleic acid molecules ofthe present invention (including those that donot themselves encode biologically active polypeptides) may be used tomap the locations of the tmst2-receptor gene and related genes onchromosomes. Mapping may be done by techniques known in the art. such asPCR amplification and in situ hybridization.

Tmst2 like nucleic acid molecules (including those that do notthemselves encode biologically active polypeptides), may be useful ashybridization probes in diagnostic assays to test, either qualitativelyor quantitatively, for the presence of an tmst2 like DNA orcorresponding RNA in mammalian tissue or bodily fluid samples.

The tmst2 like polypeptides may be used (simultaneously or sequentially)in combination with one or more cytokines, growth factors, antibiotics,anti-inflammatories, and/or chemotherapeutic agents as is appropriatefor the indication being treated.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLE 1 Isolation of Murine tmst2-Receptor Gene Using ΔkFGF-SignalTrap Method

Secretion signal trap method is a novel way to clone 5′ ends of cDNAsencoding secreted proteins from a random cDNA library. Generally, signaltrapping relies on the secretion of a reporter polypeptide by signalsequences present in a cDNA library. The secreted reporter polypeptidemay be detected by a variety of assays based upon growth selection,enzyiatic activity or immune reactivity. (See U.S. Pat. No. 5,536,637;Klein et al., Proc. Natl. Acad. Sci. USA, 93:7108-7113 (1996); Imai etal., J. Biol. Chem., 271:21514-21521 (1996)). Published PCT applicationNo. WO 96/409904 describes signal trap cloning by selection forgrowth-factor dependent cell lines and is incorporated here in byreference.

In the instant case, a novel method for trapping signal sequence DNAfrom cDNA libraries was utilized to isolate and identify novel secretedproteins, including tmst2-receptor polypeptide. In the instant case asignal trap vector kFGF7 containing a DNA molecule encoding a reporterpolypeptide that lacked a functional 5′-signal sequences was utilized ingenerating a cDNA library from a desired cell source. Secretion of thereporter polypeptide is indicative of the presence of functional signalsequence and may be detected by a variety of methods including growthunder certain conditions, enzyme activity or immune reactivity.Significantly, the molecule of the present invention, tmst2-receptorpolypeptide, was identified utilizing the method described below ofselecting signal sequences in mammalian cells (NlH 3T3 cells) by using areporter polypeptide (kFGF) which stimulated the growth of host cells.

Murine tmst2 cDNA was isolated from a mouse bone marrow stromal cellline cDNA library that was made using the signal trap kFGF7 vector,ΔkFGF7L, as previously described. Briefly, polyA⁺ RNA was prepared frommouse bone marrow stromal cell line using a commercially available RNAextraction kit (Trizol, LTI) and mRNA purification kit (Dynabeads,Dynal). The cDNA library was made according to the protocol ofSuperScript Plasmid System for cDNA synthesis and Plasmid Cloning(GIBCO/BRL, Cat. No. 18248-013) with some modification. To make cDNAwith random 3′ ends followed by a Not I site, the oligonucleotide1360-38 (SEQ ID NO: 1) was made and used as the primer for first strandcDNA synthesis.

1360-38 GGAAGGAAAAAAGCGGCCGCAACANNNNNNNNN SEQ ID NO: 1

PolyA⁺ RNA (3 μg) and 400 ng of the primer was used in the first strandreaction. After second strand synthesis using published procedures, SalI adapter ligation, and Not I digestion, double stranded cDNA was sizeselected by gel electrophoresis through 1.2% low melting agarose. Gelsection containing DNA in the 200 to 800 bp range was excised and meltedfor 10 minutes at 70° C. DNA was recovered by extraction with phenolchloroform, followed by ethanol precipitation. The recovered cDNA wasligated into vector DkFGF7L previously digested with Sall and NotI;ligation positioned the cDNA fragmients adjacent the kFGF gene lackingthe 5′-signal peptide sequence. Ligation was carried out overnight atroom temperature in a 20 μl reaction containing 50 ng vector DNA, 16 ngcDNA, 1× ligase buffer, and 1 ml of T4 ligase. The ligated DNA wasprecipitated and introduced into E.coli by electroporation as describedin the protocol.

The isolation and identification of novel cDNAs using kFGF signaltrapping method was based on the observation that NIH/3T3 cellstransfected with kFGF signal trap vectors containing test DNA fragmentsand signal sequences continued to orow and form colonies in selectionmedium while NIH/3T3 cells transfected with empty vectors oruntransfected NIH/3T3 cells did not grow in the selection medium.

Plasmid DNA from the cDNA library was prepared in pools of 50,000 colonyforming units (cfu) each. E. coli transformed with a cDNA library in theΔkFGF7L signal trap vector were plated on 150 mm LB agar plates with 100μg/ml ampicillin and incubated at 37° C. ovenight. About 50,000 colonyforming units (cfu) from agar plates were pooled into 50 ml LB in a 250ml flask. The bacteria were grown for 3 hours with agitation, andpelleted by centrifugation at 4000 rpm for 10 minutes in 50 ml conicaltubes. Ten pools were prepared. Plasmid DNA was isolated from the poolsusing QIAGEN maxi prep.

Plasmid DNA was introduced into NIH 3T3 cells by the standard calciumphosphate transfection as previously described (Sambrook et at, supra).Briefly, 100 ng of each cDNA library pool was used to transfect about200,000 cells in one 35 mm plate. After 24 hours, the cells from one 35mm plate were split into five 100 mm plates and grown in normal mediumfor one day followed by low serum medium for 13 days. About 2500colonies grew from transfected cells after the two week incubation inthe selection medium. These colonies were then analyzed for novel genesthat encoded secreted polypeptides as described below.

To each 100 mm tissue culture plate was added 2 ml of trypsin-EDTAfollowed by incubation at 37° for 5 minutes. Thecells in the colonieswere released from the surface of the plate by gentle swirling. Cellswere transferred to 50 ml conical tubes with 2 ml of FCS to stop thetrypsin activity. Tubes were centrifiged at 1000 rpm for 5 minutes topellet the cells. The supernatant was discarded.

Cells equal or less than 1 gram were lysed with 20 ml of TRIzol reagent(BRL), homogenized for 30 seconds, and extracted with 4 ml ofchloroform. The tubes were centrifuged at 4000 rpm for 3( minutes andthe aqueous phase was transferred to a new tube. RNA was precipitated byadding 10 ml isopropanol, mixing, and centrifuging, for 30 minutes at4200 rpm. The RNA pellet was washed with 10 ml of 70% ethanol, driedbriefly, and resuspended in 0.5 ml TE buffer. Total RNA from each of sixexperiments (approximately 15,000 NIH-3T3 colonies) was used to preparepolyA⁺ RNA using a commercially available mRNA purification kit (Dynal).The cDNA inserts of the plasmid transcripts were rescued by RI-PCR. ASuperScript preamplification system (BRL) was used to synthesize firststrand cDNA. For each reaction representing one of six experiments, 1 μgpolyA⁺ RNA, 1 μl (2 mM) vector-specific primer 1605-21 (SEQ ID NO: 2),and water were combined in a total volume of 12 μl.

1605-21 5′AATCCGATGCCCACGTTGCAGTA 3′ SEQ ID NO: 2

The mixture was incubated at 70° C. for 10 minutes and transferred to50° C. A premixture was prepared containing 2.0 μl 10×buffer, 2.0 μl of25 mM MgCl₂, 1.0 μl 10 mM dNTPs, and 2.0 μl 0.1 M dithiothreitol wasadded. The reaction was started by the addition of 1.0 ml reversetranscriptasc and incubated at 50° C. for one hour. The reaction wasstopped by incubation at 70° C. for 15 minutes. The RNA was digestedwith 1 μl Rnase H at 37° C. for 20 minutes.

PCR was perfomied with Pfu polymerase (Perkin Elmer). In a total volumeof 100 μl, 2 μl first strand reaction, 1×Pfu buffer, 0.4 μM each ofprimers 1239-08 (SEQ ID NO: 3) and 1605-22 (SEQ ID NO: 4), 0.2 mM dNTPs,5% DMSO, and 1.0 μl Pfu polymerase were added.

1239-08: 5′ AAAATCTTAGACCGACGACTGTGTTT 3′ SEQ ID NO: 3

1605-22: 5′ GAGTCTCCGCAGCCTTTTGAGG SEQ ID NO: 4

The sample was heated at 95° C. for 1 minute, and amplified for 30cycles. Each cycle includes: 95° C. for 30 seconds, 66° C. for 45seconds, 72° C. for 2 minutes. The reaction was incubated at 70° C. for10 minutes at the end.

PCR DNA fragments were extracted once with phenol/chloroform (50/50) andethanol precipitated. The DNA was then digested with NotI and Sall andsmall fragments and PCR primers were removed by using a QIAGEN PCRpurification kit (QIAGEN). A signal trap library was constructed byligating the DNA fragments into XhoI and Not I digested vector,pcDNA3.(−) containing the placental alkaline phosphatase gene. Eachligation included 10 ng PCR fragments, 50 ng vector, 1×ligase buffer,and 0.5 μl T4 DNA ligase in a total volume of 10 μl. The ligation wascarried at 16° C. overnight. The ligated DNA was precipitated by adding5 μl tRNA, 10 μl water, 12.5 μl 7.5 M NH⁴AC, 70 μl ethanol (−20° C.),and centrifuged for 20 minutes. The pellet was washed with 0.5 ml 70%ethanol (−20° C.), and resuspended in 5 μl water. For each of the sixligation reactions, 8 μl was used to transform 100 μl of E. coli DH10Bcells by electroporation. A total of 36 million cfu was obtained.

One clone, tmst2-00004-d1, was found to contain a 412 nucleotide insert(SEQ ID NO. 5) encoding the amino terminal 133 amino acids (SEQ ID NO.6) of the full-length protein including the signal peptide.

EXAMPLE 2 DNA Encoding Full Length Mouse tmst2 Receptor

A cDNA encoding the full length mouse tmst2 receptor was constructed bycombining two sequences. The first sequence was the original signal trapclone. The second sequence was obtained by screening a cDNA library fromthe bone marrow stromal cell line, using thc 412 bp signal trap clone asa probe, following standard colony hybridization procedure (Sambrook elal.). The longest clone obtained from the screen encoded the full lengthprotein except for the five amino terminal amino acids. The clone alsoincluded 16 nucleotides of 3′ untranslated region (3′ UTR) followed by apoly A stretch. An alternative downstream poly A addition signal wasdeduced from additional poly-adenylated 3′ UTR sequence obtained by 3′RACE using a Marathon cDNA library from a seven day seven mouse embryo(Clontech). The full-length sequence (SEQ ID NO: 7) includes an openreading framne of 594 nucleotides encoding a primary translation productof 198 amino acids (SEQ ID NO: 8) having a predicted size ofapproximately 20 kD. The deduced protein sequence has a predicted aminoterminal signal peptide and a carboxy terminal transmembrane domainfollowed by a dibasic stop transfer signal. Comparison ofthe deducedamino acid sequence (SEQ ID NO. 8) and predicted disulfide linkagestructure of tmst2 with those of other members of the TNF-receptor genefamily reveals that tmst2 is most closely related to ymkz5 (a novel UNFreceptor cloned at Amgen), FAS and TNFR-1.

Alternative polyadenylation sites yield 3′ UTR's of either 16 or 26nucleotides. The longer 3′ UTR harbors a repeated ATTT sequence. Thismotif has been implicated in RNA stability and translational control,suggesting that tmst2 expression may be under control of externalstimuli. The 3′ RACE clones also showed the existence of a splicevariant (SEQ ID NO. 9) in which a 45 bp alternative exon is insertedbetween nucleotides 523 and 524 of the “transmembrane” full lengthsequence (SEQ ID NO: 7). The inserted sequence causes translationaltermination before the transmembrane domain and the resultingvariantprotein (SEQ ID NO: 10) is predicted to be secreted.

EXAMPLE 3 Tissue Specific Expression of tmst2-Receptor

Tissue specific expression patterns of tmst2-receptor gene wasinvestigated by Northern blot analysis and itn situ hybridization usinga ³²P-labeled PCR product as a probe to detect the presence oftmst2-receptor transcript in various tissues.

Cytoplasmic and poly-A⁺ RNA were isolated from various cell lines andtissues using standard techniques [Sambrook, J. et al, MolecularCloning, Cold Spring Harbor Laboratory Press, New York (1989)].Cells/tissues were lysed with 20 ml of TRIzol reagent (BRL), homogenizedfor 30 seconds, and extracted with 4 ml of chloroform. The tubes werecentrifuged at 4000 rpm for 30 minutes and the aqueous phase wastransferred to a new tube. RNA was precipitated by adding 10 mlisopropanol, mixing, and centrifuging for 30 minutes at 4200 rpm. TheRNA pellet was washed with 10 ml of 70% ethanol, dried briefly, andresuspended in 0.5 ml TE buffer. Poly A⁺ RNA was prepared by using acommercially available mRNA purification kit (Dynal).

After elution of poly A⁺ RNA from the column in 750 μl of TE buffer, thesample was then ethanol precipitated by adding 40 μl sample buffer and 1ml ethanol at −70° C. overnight. Poly A⁺ RNA was then fractionated usingformaldehlyde/agarose gel electrophioresis system as previouslydescribed and transferred. Following electrophoresis, the gel wasprocessed and the .RNA transferred to a nylon membrane. See Sambrook etal. Supra. Commercially available RNA blots (Clontech) were also used.Northern blots were then prehybridized in 20 ml of prehybridizationsolution containing 5×SSPE, 50% fomarnide, 5×Denhardt's solution, 0.5%SDS and 100 mg/mil denatured salmon spenn DNA for 2-4 hours at 42° C.The blots were then hybridized in 20 ml of hybridization solutioncontaining 6×SSPE, 50% formamide, 5×Denhardt's solution, 0.5% SDS, 100μg/ml denatured salmon sperm DNA. Approximately 5 ng/ml of randomprimed, ³²P-labeled (RadPrime Kit, GIBCO) tmst2-00004-d1 cDNA was usedas a probe. The blots were hybridized for 18-24 hours at 42° C. Theblots were then washed in 2×SSC, 0.1 % SDS at 42° C. The blots were thenexposed to x-ray films for three days at 80° C.

Northern blot analysis revealed that tmst2 gene is expressed in early (7day) embryo's, NIH-3T3 cells, and bone marrow stromal cells. Low leveltmst2 transcripts were also detected in 7-day embryos, and in adultheart, lungs, small intestines and kidneys by in situ hybridization.

In situ hybridization analysis is carried out to detect the presence anddistribution of mRNA in different tissues. Iz situ hybridization iscarried out as previously described. See Sambrook et al., supra.Briefly, a panel of normal embryonic (E8.5 through E15.5) and adultmouse tissues are fixed in zinc-formalin fixative, embedded in paraffin,and sectioned to generate 5 μm thin sections. Following sectioning andprior to in situ hybridization, tissue sections were permeabilized with0.2 M HCl, followed by digestion with Proteinasc K. The sections areacetylated with triethanolamine and acetic anhydride. Sections arehybridized overnight at 55° C. with a ³²P-labelcd riboprobecorresponding to the full length mouse cDNA that was generated using thestandard protocols was for example pGEM vectors having RNA polyncrasetranscription initiation sites. Excess probe is removed by RNasedigestion followed by a series of washes in buffer with decreasing saltconcentrations followed by a high stringent wash in 0.1×SSC at 55° C.The sections are then processed for autoradiography. The sections aredipped in Kodak NTB2 photographic emulsion, and kept a 4° C. forapproximately 2-3 weeks. The sections are then developed andcounterstained with hematoxilyn and eosin. Sections were examined usingdarkficld and transmitted light microscopy for tissue morphololy andhybridization signals.

EXAMPLE 4 Production of tmst2-Receptor Polypeptides

A. Expression of tmst2-Receptor Polypeptide in Bacteria

PCR may be used to amplify template DNA sequences encoding antmst2-receptor polypeptide using primers corresponding to the 5′ and 3′ends of the sequence. The amplified DNA products may be modified tocontain restriction enzyme sites to allow for insertion into expressionvectors. PCR products are gel purified and inserted into expressionvectors using standard recombinant DNA methodology. An exemplary vector,such as pAMG21 containing the lux promoter and a gene encodingikanamycin resistance is digested with BamHI and NdcI for directionalcloning of inserted DNA. The ligated mixture is transformed into E. colihost strain 393 by electroporation and transformants selected forkanamycin resistance. Plasmid DNA from selected colonies is isolated andsubjected to DNA sequencing to confirm the presence of the insert.

Transformed host cells are incubated in 2XYT medium containing 30 μg/mlkanamycin at 30° C. prior to induction. Gene expression can then beinduced by addition of N-(3-oxohexanoyl)-d1-homoserine lactone to afinal concentration of 30 ng/ml followed by incubation at either 30° C.or 37° C. for six hours. Expression of tmst2-receptor polypeptide isevaluated by centrifugation of the culture, resuspension and lysis ofthe bacterial pellets, and analysis of host cell proteins bySDS-polyacrylamide gel electrophoresis.

According to the protocol above, secreted tmst2 (SEQ ID NO: 10) wasproduced in E. coli. The purified protein of approximately 20 kD wasthen used for biological studies and antibody production.

Inclusion bodies containing tmst2-receptor polypeptide are purified asfollows: Bacterial cells are pelleted by centrifugation and resuspendedin water. The cell suspension is lysed by sonication and pelleted bycentrifugation at 195,000×g for 5 to 10 minutes. The supernatant isdiscarded and the pellet washed and transferred to a homogenizer. Thepellet is homo,enized in 5 ml. of a Percoll solution (75% liquidPercoll. 0.15 M NaCl) until uniformly suspended and then diluted andcentrifuged at 21,600×g for 30 minutes. Gradient fractions containingthe inclusion bodies are recovered and pooled. The isolated inclusionbodies are analyzed by SDS-PAGE.

B. Expression of tmst2-Receptor Polypeptide in Mammalian Cells

A cDNA fragment encoding the 171 amino terminal residues of tmst2 wasprepared using PCR. Briefly, the tmst2 cDNA region was amplified usingprimers 2086′-39 and 2086-41 corresponding to the 5′ and 3′-ends of thecDNA.

2086-39 CATACTAGTTCCACCATGTTTGGCTTCTTCTGCAGCTTGGT SEQ ID NO: 11

2086-41 TTGTCGACATTTGAAACAGATGAACTGCACACA SEQ ID NO: 12

The resulting fragment was digested with Spel and Sal I, and ligatedinto the XbaI and SalI sites of pDSRaFc plasmid vector containing a DNAinsert encoding the human Fc region. The resulting fusion gene wasconfirmed by DNA sequencing (SEQ ID NO: 13). The deduced amino acidsequence of the tmst2-Fc fusion protein is set out in SEQ ID NO: 14.

The expression constrict was transfected into CHOD-AM1 cells by calciumphosphate method as previously described (Ausubel et al., Curr. Prot.Mol. Biol. 1, 9.11-9.13, 1994) and transfected cells were selected indialyzed serum in the absence of HT supplement. Individual colonies wereexpanded and conditioned medium was tested for fusion protein productionby Western analysis. A 55 kD band representing the fusion protein wasobserved at varying levels. One clone producing approximately I mg ofFc-fusion protein per liter was adapted to suspension growth and usedfor large-scale production.

EXAMPLE 5 Production of Anti-tmst2-receptor Antibodies

Antibodies to tmst2-receptor polypeptides may be obtained byimmunization with purified protein or with tmst2-receptor peptidesproduced by biological or chemical synthesis. Substantially pure tmst2protein or polypeptide may be isolated from trans fcted cells asdescribed in Example 4. Concentration of protein in the finalpreparation may be adjusted, for example, by concentration oni an amiconfilter device, to the level of a few micrograms/ml. Monoclonal orpolyclonal antibodies to the protein can then be prepared by any of theprocedures known in the art for generating antibodies such as thosedescribed in Hudson and Bay, “Practical Immunology, Second Edition”,Blackwell Scientific Publications.

A. Anti-tmst2-receptor Monoclonal Antibody Production

Monoclonal antibody to epitope of any of the peptides identified andisolated as described can be prepared from murine hybridomas accordingto the classical method of Kohler, G. and Milstein, C., Nature 256:495(1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein over a periodof a few weeks. The mouse is then sacrificed, and the antibody producingcells of the spleen isolated. The spleen cells are fused by means ofpolyethylene glycol with mouse myeloma cells such as NS-1 cells, and theexcess unfused cells destroyed by growth of the system on selectivemedia comprising hypoxanthine; aminopterin; thynidine (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.After selection, tissue culture supernatants are taken from each fusionwell and tested for tmst2-receptor antibody production by EIA. Selectedpositive clones can be expanded and their monoclonal antibody productharvested for use. Detailed procedures for monoclonal antibodyproduction are described in Davis, L. et al. Basic Methods in MolecularBiology, Section 21-2, Elsevier, New York, N.Y.

B. Polyclonal Anti-tmst2 Receptor Antibody Production

Polyclonal antiserum containing antibodies to heterogenous epitopes of asingle protein can be prepared by immunizing suitable animals with theexpressed protein described above, which can be unmodified or modifiedto enhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanlarge molecules and may require the use of carriers or adjuvants. Also,lost animals vary in response to site of inoculations and dose, withboth inadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng levels) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin.Endocrinol. Metab. 33: 988-991 (1971).

Booster injections can be given at regrular intervals, and antiserumharvested when antibody titer thereof as determined semi-quantitativcly,(or example, by double immunodiffosion in agar against knownconcentrations of the antigen, begin to fall. See, for example,Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunologyed. D. Weir, Blackwell (1973). Plateau concentration of antibody isusually in the range of 0.1 to 0.2 mg/ml of serim (about 12 um).Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,Chapt. 42 in; Manual of Clinical Immunology, 2d Ed. (Rose and Friedman,eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).

Three rabbits were immunized with tmst2 protein produced in E. coli.Test bleeds show that the serum of all rabbits will immunoprecipitatethe tmst2 protein as well as detect it on Western blots.

Alteniative procedures for obtaining anti-tmst2-receptor antibodies mayalso bc employed, such as immunization of transgenic mice harboringhuman Ig loci for production of fully human antibodies, and screening ofsynthetic antibody libraries, such as those generated by mutagenesis ofan antibody variable domain.

EXAMPLE 6 Biological Activity of tmst2-Receptor Polypeptide

Secreted tmst2 receptor (SEQ ID NO. 10) was expressed in E.coli asdescribed above and tested in vitro for the ability to bild to andneutralize the activity of members of the TNF-related ligand family.

At 10 mg/ml and 100 mg/ml, secreted murine tmst2 blocked apoptosis inJurkat cells induced by murine TRAIL protein. However, secreted murinetmst2 failed to block apoptosis in Jurkat cells when induced by eitherhuman TRAIL or by human FAS ligand. These results indicate that secretedmurine tmst2 is biologically active and may be involved in regulatingthe activity of members of the TNF family including 1TRAIL.

EXAMPLE 7 Functional Analysis of the Role of tmst2-Receptor

To determine the functional role of tmst2 in vivo, the tmst2 gene iseither over expressed in the germ line of animals or inactivated in thegerm line to mammals by homologous recombination. Animals in which thegene is over expressed under the regulatory control of exogenous orendogenous promoter elements are known as transgenic animals. Animals inwhich an endogenous gene has been inactivated by homologousrecombination are also known as “knockout” animals. Exemplary mammalsinclude rabbits and rodent species such as mice.

Transgenic animals allow for the determination of the effect(s) of overexpression or inappropriate expression of the tmst2-receptor ondevelopment and disease processes. tmst2-receptor-receptor transglenicanimals can also serve as a model system to test compounds that canmodulate receptor activity.

The “knockout” animals allow for the determination of the role of tmst2in embryonic development, and in immune and proliferative responses. Therole of tmst2 in development, and in immune and proliferative responseis determined by analysis the effect(s) of gene knockout on thedevelopment of the embryo as well as on the development anddifferentiation of various organs and tissues such as the immune systemin these animals. (as determined by FACS analysis ofcell populations atdifferent stages of development).

In addition to tmst2 “knockout” mammals, double and triple “knockout” oftmst2 and one or two related genes are contemplated.

EXAMPLE 8 tmst2-receptor-Related Genes

Southern analysis of mouse genomic DNA was carried out using standardhybridization conditions and final washes at 0.1×SSC, 0.1% SDS at 42° C.After digestion with each of the following enzyines: EcoRI, BamHI, PstI,HindIII and EcoRV, three restriction fragments were shown to hybridizeto a tmst2 cDNA probe. This observation suggested the existence of oneor more genes related to tmst2, and the EcoRI fragments of 7.5, 6.0 and4.5 kB were cloned in a bacteriophage 1 vector. The cloned genomicsequences related to tmst2 are used to identify human homologs.

EXAMPLE 9 Binding Analysis of TNF Ligand Family Members withtmst2-Receptor

Binding studies were performed to determine if various TNF ligand familymembers are ligands for the tmst2-receptor. The only TNF ligand whichbound to the tmst2-receptor was murine TRAIL. The binding studies, basedon surface plasmon resonance, were carried out with the automated, highthroughput Biacore 2000 system at 25° C. according to the manufacturer'sinstructions (Biacore, Uppsula, Sweden) as follows:

The receptor was immobilized on a CM5 research grade amine coupling chip(Biacore) by placing 40 μg/ml of recombinant tmst-2 receptor diluted inHEPES buffered saline (HBS-P; Biacore) at pH 4.5 on the chip. Theligands were diluted in HBS-P buffer containing 10 μg/ml BSA and 4 mg/mldextran to block nonspecific binding sites. The ligand samples wereinjected over the receptors at concentrations ranging from 2 nM to 100nM. The chips were regenerated between ligand injections by washing 2times for 3 minutes in 25 mM CAPS, 1 M NaCl pH 10.5.

The TNF ligands tested were: human nag TRAIL,(amino acids 95-281;Genbank accession no. AAC50332), murine nag TRAIL (amino acids 99-29,Genbank accession no. NP 033451), murine Fc TRAIL, human OPGL ligand(amino acids 159-318), murine OPGL ligand (amino acids 159-316) humanTNFα (amino acids 82-233, Genbank accession no. CAA26669), murine TNFα(amino acids 82-233, Genbank accession no. CAA68530). The extracellularand transmembrane portions of the ligands were recombinantly expressedin E.coli. Specifically, the denoted amino acids for each ligandindicate the portion of the ligand expressed recombinantly.Additionally, commercially available human Fas ligand (AlexisBiochemicals, San Diego, Calif.) was also tested. Human DR5Fc, a knownTRAIL receptor, was used as a control.

Results of the assay were determined by detecting the change in mass onthe chip as measured by changes in light absorption on the chipindicated as resonance units. The tmst2-receptor only bound to muriineTRAIL, which was bioactive in cell culture assays. The species specificbinding suggests that tmst2 polypeptide may function as a TRAIL decoyreceptor.

Similar species specific bindings to TRAIL was demonstrated for ynkz5, anovel transmembranc TNF receptor. cloned by Amgen, which is closelylinked to tmst2 in the murine genome. Primary sequences homologycomparisons indicate that both tmst2 and ymkz5 are most closely relatedto FAS and TNFR-1 which are not functionally similar. Therefore, thecharacterization of the murine genes, tmst2 and ymkz5, may aid in thediscovery of human TRAIL decoy receptors based on functionality and notsolely based on primary sequence homology.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

15 1 33 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 ggaaggaaaa aagcggccgc aacannnnnn nnn 33 2 23 DNA ArtificialSequence Description of Artificial Sequence primer 2 aatccgatgcccacgttgca gta 23 3 26 DNA Artificial Sequence Description of ArtificialSequence primer 3 aaaatcttag accgacgact gtgttt 26 4 22 DNA ArtificialSequence Description of Artificial Sequence primer 4 gagtctccgcagccttttga gg 22 5 412 DNA Mus musculus CDS (13)..(411) tmst2 00004-d1 5ttgcactcgg cc atg ttt ggc ttc ttc tgc agc ttg gtg tcc agt ctg agt 51 MetPhe Gly Phe Phe Cys Ser Leu Val Ser Ser Leu Ser 1 5 10 cgc tgg ttc ctttgg cgg cgg ctg ctg ctg ctg ctg ctg ctg ctg ctg 99 Arg Trp Phe Leu TrpArg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu 15 20 25 ctg aat ctg ccc ttgcag gta aaa ttt gct atg cta gaa tta cac tcc 147 Leu Asn Leu Pro Leu GlnVal Lys Phe Ala Met Leu Glu Leu His Ser 30 35 40 45 ttc aaa tgt ccc gctggt gaa tac tgg tct aaa gac gtc tgt tgc aag 195 Phe Lys Cys Pro Ala GlyGlu Tyr Trp Ser Lys Asp Val Cys Cys Lys 50 55 60 aac tgt tct gca ggt acattt gtc aag gcg ccc tgc gaa atc ccc cat 243 Asn Cys Ser Ala Gly Thr PheVal Lys Ala Pro Cys Glu Ile Pro His 65 70 75 act caa gga caa tgt gag aagtgt cac cca gga aca ttc aca gag aaa 291 Thr Gln Gly Gln Cys Glu Lys CysHis Pro Gly Thr Phe Thr Glu Lys 80 85 90 gat aat tac ctg gat gct tgt atactt tgc tcc acc tgt gat aaa gat 339 Asp Asn Tyr Leu Asp Ala Cys Ile LeuCys Ser Thr Cys Asp Lys Asp 95 100 105 cag gaa atg gtg gcc gac tgc tcagcc acc agt gac cgg aaa tgc cag 387 Gln Glu Met Val Ala Asp Cys Ser AlaThr Ser Asp Arg Lys Cys Gln 110 115 120 125 tgc cga aca ggt ctt tac tactat g 412 Cys Arg Thr Gly Leu Tyr Tyr Tyr 130 6 133 PRT Mus musculus 6Met Phe Gly Phe Phe Cys Ser Leu Val Ser Ser Leu Ser Arg Trp Phe 1 5 1015 Leu Trp Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Asn Leu 20 2530 Pro Leu Gln Val Lys Phe Ala Met Leu Glu Leu His Ser Phe Lys Cys 35 4045 Pro Ala Gly Glu Tyr Trp Ser Lys Asp Val Cys Cys Lys Asn Cys Ser 50 5560 Ala Gly Thr Phe Val Lys Ala Pro Cys Glu Ile Pro His Thr Gln Gly 65 7075 80 Gln Cys Glu Lys Cys His Pro Gly Thr Phe Thr Glu Lys Asp Asn Tyr 8590 95 Leu Asp Ala Cys Ile Leu Cys Ser Thr Cys Asp Lys Asp Gln Glu Met100 105 110 Val Ala Asp Cys Ser Ala Thr Ser Asp Arg Lys Cys Gln Cys ArgThr 115 120 125 Gly Leu Tyr Tyr Tyr 130 7 1550 DNA Mus musculus CDS(13)..(606) mouse tmst2 7 ttgcactcgg cc atg ttt ggc ttc ttc tgc agc ttggtg tcc agt ctg agt 51 Met Phe Gly Phe Phe Cys Ser Leu Val Ser Ser LeuSer 1 5 10 cgc tgg ttc ctt tgg cgg cgg ctg ctg ctg ctg ctg ctg ctg ctgctg 99 Arg Trp Phe Leu Trp Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu15 20 25 ctg aat ctg ccc ttg cag gta aaa ttt gct atg cta gaa tta cac tcc147 Leu Asn Leu Pro Leu Gln Val Lys Phe Ala Met Leu Glu Leu His Ser 3035 40 45 ttc aaa tgt ccc gct ggt gaa tac tgg tct aaa gac gtc tgt tgc aag195 Phe Lys Cys Pro Ala Gly Glu Tyr Trp Ser Lys Asp Val Cys Cys Lys 5055 60 aac tgt tct gca ggt aca ttt gtc aag gcg ccc tgc gaa atc ccc cat243 Asn Cys Ser Ala Gly Thr Phe Val Lys Ala Pro Cys Glu Ile Pro His 6570 75 act caa gga caa tgt gag aag tgt cac cca gga aca ttc aca gag aaa291 Thr Gln Gly Gln Cys Glu Lys Cys His Pro Gly Thr Phe Thr Glu Lys 8085 90 gat aat tac ctg gat gct tgt ata ctt tgc tcc acc tgt gat aaa gat339 Asp Asn Tyr Leu Asp Ala Cys Ile Leu Cys Ser Thr Cys Asp Lys Asp 95100 105 cag gaa atg gtg gcc gac tgc tca gcc acc agt gac cgg aaa tgc cag387 Gln Glu Met Val Ala Asp Cys Ser Ala Thr Ser Asp Arg Lys Cys Gln 110115 120 125 tgc cga aca ggt ctt tac tac tat gac cca aaa ttt cca gaa tcgtgc 435 Cys Arg Thr Gly Leu Tyr Tyr Tyr Asp Pro Lys Phe Pro Glu Ser Cys130 135 140 cgc cca tgt acc aag tgt ccc caa gga atc cct gtc ctc cag gaatgc 483 Arg Pro Cys Thr Lys Cys Pro Gln Gly Ile Pro Val Leu Gln Glu Cys145 150 155 aac tcc aca gct aac act gtg tgc agt tca tct gtt tca aat cccaga 531 Asn Ser Thr Ala Asn Thr Val Cys Ser Ser Ser Val Ser Asn Pro Arg160 165 170 aac cgg ctg ttc cta ctg tta tca cct ttg agt gtg cta att gtgtcc 579 Asn Arg Leu Phe Leu Leu Leu Ser Pro Leu Ser Val Leu Ile Val Ser175 180 185 gtt gtt gtc ttc cgt atc ata aga aga taaaggttct acagatgttt626 Val Val Val Phe Arg Ile Ile Arg Arg 190 195 tcttagcttc cttttattgctatgaagtga tactatggag gcaactcttt tattttattt 686 attttatttt attttttaatgtcttgaact tgatttgaag accaggctgg cctcaaaatc 746 acagagatcc agactaagacaactctaata agggaaacat ttaattggga ctggcttaca 806 gtttcggacg ttttgtccatgattatcata gtgggaagca tggcagcatc taagcagaca 866 tgatgttgga gaaggagctgagatttctgc atcttgatct gcaagcaata aaaggagact 926 gtgtgccaca ctatacacagcttgaacata ggagacctca aagcctgtcc ccacagtgac 986 aaacttcctc caacaaggtcatacctccta ataataccat ttcttatgag gcaagcattc 1046 aaacacatga gtctatgagggccaaaccaa ttcaaaccac cacaggttaa caattgccct 1106 ctgcagctct ctggtggaggccctccttga gagtaagtaa caatttagat gaaggcaagt 1166 cctggtatca ggtccaaaagaaactcagga tgaatggtcc actgtggttc ctattaacat 1226 actgaagaac atgacctcaccttacacgtc tccacctcac tgacttccct tcccctagct 1286 tctcattccc aggtaaccctgccatttttt ggtaatgtgc cttcttggtt cttcctctcc 1346 tttccccctc tcttctggtccttacttctc ttcctctccc actctccacc agcctcctct 1406 taaggcctga atcagtctgtaggtcatgtt taatctacta ctttctctct gctctggact 1466 catccagatg tctctggctgagctctccct cctatctaca ataaaaccct tccccctaac 1526 cagaaatgca aaaaaaaaaaaaaa 1550 8 198 PRT Mus musculus 8 Met Phe Gly Phe Phe Cys Ser Leu ValSer Ser Leu Ser Arg Trp Phe 1 5 10 15 Leu Trp Arg Arg Leu Leu Leu LeuLeu Leu Leu Leu Leu Leu Asn Leu 20 25 30 Pro Leu Gln Val Lys Phe Ala MetLeu Glu Leu His Ser Phe Lys Cys 35 40 45 Pro Ala Gly Glu Tyr Trp Ser LysAsp Val Cys Cys Lys Asn Cys Ser 50 55 60 Ala Gly Thr Phe Val Lys Ala ProCys Glu Ile Pro His Thr Gln Gly 65 70 75 80 Gln Cys Glu Lys Cys His ProGly Thr Phe Thr Glu Lys Asp Asn Tyr 85 90 95 Leu Asp Ala Cys Ile Leu CysSer Thr Cys Asp Lys Asp Gln Glu Met 100 105 110 Val Ala Asp Cys Ser AlaThr Ser Asp Arg Lys Cys Gln Cys Arg Thr 115 120 125 Gly Leu Tyr Tyr TyrAsp Pro Lys Phe Pro Glu Ser Cys Arg Pro Cys 130 135 140 Thr Lys Cys ProGln Gly Ile Pro Val Leu Gln Glu Cys Asn Ser Thr 145 150 155 160 Ala AsnThr Val Cys Ser Ser Ser Val Ser Asn Pro Arg Asn Arg Leu 165 170 175 PheLeu Leu Leu Ser Pro Leu Ser Val Leu Ile Val Ser Val Val Val 180 185 190Phe Arg Ile Ile Arg Arg 195 9 702 DNA Mus musculus CDS (13)..(552)Primer 2086-39 9 ttgcactcgg cc atg ttt ggc ttc ttc tgc agc ttg gtg tccagt ctg agt 51 Met Phe Gly Phe Phe Cys Ser Leu Val Ser Ser Leu Ser 1 510 cgc tgg ttc ctt tgg cgg cgg ctg ctg ctg ctg ctg ctg ctg ctg ctg 99Arg Trp Phe Leu Trp Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu 15 20 25ctg aat ctg ccc ttg cag gta aaa ttt gct atg cta gaa tta cac tcc 147 LeuAsn Leu Pro Leu Gln Val Lys Phe Ala Met Leu Glu Leu His Ser 30 35 40 45ttc aaa tgt ccc gct ggt gaa tac tgg tct aaa gac gtc tgt tgc aag 195 PheLys Cys Pro Ala Gly Glu Tyr Trp Ser Lys Asp Val Cys Cys Lys 50 55 60 aactgt tct gca ggt aca ttt gtc aag gcg ccc tgc gaa atc ccc cat 243 Asn CysSer Ala Gly Thr Phe Val Lys Ala Pro Cys Glu Ile Pro His 65 70 75 act caagga caa tgt gag aag tgt cac cca gga aca ttc aca gag aaa 291 Thr Gln GlyGln Cys Glu Lys Cys His Pro Gly Thr Phe Thr Glu Lys 80 85 90 gat aat tacctg gat gct tgt ata ctt tgc tcc acc tgt gat aaa gat 339 Asp Asn Tyr LeuAsp Ala Cys Ile Leu Cys Ser Thr Cys Asp Lys Asp 95 100 105 cag gaa atggtg gcc gac tgc tca gcc acc agt gac cgg aaa tgc cag 387 Gln Glu Met ValAla Asp Cys Ser Ala Thr Ser Asp Arg Lys Cys Gln 110 115 120 125 tgc cgaaca ggt ctt tac tac tat gac cca aaa ttt cca gaa tcg tgc 435 Cys Arg ThrGly Leu Tyr Tyr Tyr Asp Pro Lys Phe Pro Glu Ser Cys 130 135 140 cgc ccatgt acc aag tgt ccc caa gga atc cct gtc ctc cag gaa tgc 483 Arg Pro CysThr Lys Cys Pro Gln Gly Ile Pro Val Leu Gln Glu Cys 145 150 155 aac tccaca gct aac act gtg tgc agt tca tct gtt tca aga aga tct 531 Asn Ser ThrAla Asn Thr Val Cys Ser Ser Ser Val Ser Arg Arg Ser 160 165 170 gcc tcagtg gcc tgg cct atc tgaatggttc acagagatcc cagaaaccgg 582 Ala Ser Val AlaTrp Pro Ile 175 180 ctgttcctac tgttatcacc tttgagtgtg ctaattgtgtccgttgttgt cttccgtatc 642 ataagaagat aaaggttcta cagatgtttt cttagcttccttttattgct atgaagtgat 702 10 180 PRT Mus musculus 10 Met Phe Gly Phe PheCys Ser Leu Val Ser Ser Leu Ser Arg Trp Phe 1 5 10 15 Leu Trp Arg ArgLeu Leu Leu Leu Leu Leu Leu Leu Leu Leu Asn Leu 20 25 30 Pro Leu Gln ValLys Phe Ala Met Leu Glu Leu His Ser Phe Lys Cys 35 40 45 Pro Ala Gly GluTyr Trp Ser Lys Asp Val Cys Cys Lys Asn Cys Ser 50 55 60 Ala Gly Thr PheVal Lys Ala Pro Cys Glu Ile Pro His Thr Gln Gly 65 70 75 80 Gln Cys GluLys Cys His Pro Gly Thr Phe Thr Glu Lys Asp Asn Tyr 85 90 95 Leu Asp AlaCys Ile Leu Cys Ser Thr Cys Asp Lys Asp Gln Glu Met 100 105 110 Val AlaAsp Cys Ser Ala Thr Ser Asp Arg Lys Cys Gln Cys Arg Thr 115 120 125 GlyLeu Tyr Tyr Tyr Asp Pro Lys Phe Pro Glu Ser Cys Arg Pro Cys 130 135 140Thr Lys Cys Pro Gln Gly Ile Pro Val Leu Gln Glu Cys Asn Ser Thr 145 150155 160 Ala Asn Thr Val Cys Ser Ser Ser Val Ser Arg Arg Ser Ala Ser Val165 170 175 Ala Trp Pro Ile 180 11 41 DNA Artificial SequenceDescription of Artificial Sequence primer 11 catactagtt ccaccatgtttggcttcttc tgcagcttgg t 41 12 33 DNA Artificial Sequence Description ofArtificial Sequence primer 12 ttgtcgacat ttgaaacaga tgaactgcac aca 33 131200 DNA Artificial Sequence Description of Artificial Sequence Fusionprotein consisting of Mus musculus sequences and Immunoglobulinsequences 13 atg ttt ggc ttc ttc tgc agc ttg gtg tcc agt ctg agt cgc tggttc 48 Met Phe Gly Phe Phe Cys Ser Leu Val Ser Ser Leu Ser Arg Trp Phe 15 10 15 ctt tgg cgg cgg ctg ctg ctg ctg ctg ctg ctg ctg ctg ctg aat ctg96 Leu Trp Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Asn Leu 20 2530 ccc ttg cag gta aaa ttt gct atg cta gaa tta cac tcc ttc aaa tgt 144Pro Leu Gln Val Lys Phe Ala Met Leu Glu Leu His Ser Phe Lys Cys 35 40 45ccc gct ggt gaa tac tgg tct aaa gac gtc tgt tgc aag aac tgt tct 192 ProAla Gly Glu Tyr Trp Ser Lys Asp Val Cys Cys Lys Asn Cys Ser 50 55 60 gcaggt aca ttt gtc aag gcg ccc tgc gaa atc ccc cat act caa gga 240 Ala GlyThr Phe Val Lys Ala Pro Cys Glu Ile Pro His Thr Gln Gly 65 70 75 80 caatgt gag aag tgt cac cca gga aca ttc aca gag aaa gat aat tac 288 Gln CysGlu Lys Cys His Pro Gly Thr Phe Thr Glu Lys Asp Asn Tyr 85 90 95 ctg gatgct tgt ata ctt tgc tcc acc tgt gat aaa gat cag gaa atg 336 Leu Asp AlaCys Ile Leu Cys Ser Thr Cys Asp Lys Asp Gln Glu Met 100 105 110 gtg gccgac tgc tca gcc acc agt gac cgg aaa tgc cag tgc cga aca 384 Val Ala AspCys Ser Ala Thr Ser Asp Arg Lys Cys Gln Cys Arg Thr 115 120 125 ggt ctttac tac tat gac cca aaa ttt cca gaa tcg tgc cgc cca tgt 432 Gly Leu TyrTyr Tyr Asp Pro Lys Phe Pro Glu Ser Cys Arg Pro Cys 130 135 140 acc aagtgt ccc caa gga atc cct gtc ctc cag gaa tgc aac tcc aca 480 Thr Lys CysPro Gln Gly Ile Pro Val Leu Gln Glu Cys Asn Ser Thr 145 150 155 160 gctaac act gtg tgc agt tca tct gtt tca aat gtc gac act cac aca 528 Ala AsnThr Val Cys Ser Ser Ser Val Ser Asn Val Asp Thr His Thr 165 170 175 tgccca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc 576 Cys ProPro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 180 185 190 ctcttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 624 Leu PhePro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 195 200 205 gaggtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 672 Glu ValThr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 210 215 220 aagttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 720 Lys PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 225 230 235 240aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 768 LysPro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 245 250 255ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 816 LeuThr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 260 265 270aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc 864 LysVal Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 275 280 285aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 912 LysAla Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 290 295 300tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc 960 SerArg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 305 310 315320 aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 1008Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 325 330335 cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 1056Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 340 345350 ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 1104Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 355 360365 cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 1152Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 370 375380 aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tgataa 1200Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 14398 PRT Artificial Sequence Description of Artificial Sequence Fusionprotein consisting of Mus musculus sequences and Immunoglobulinsequences 14 Met Phe Gly Phe Phe Cys Ser Leu Val Ser Ser Leu Ser Arg TrpPhe 1 5 10 15 Leu Trp Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu LeuAsn Leu 20 25 30 Pro Leu Gln Val Lys Phe Ala Met Leu Glu Leu His Ser PheLys Cys 35 40 45 Pro Ala Gly Glu Tyr Trp Ser Lys Asp Val Cys Cys Lys AsnCys Ser 50 55 60 Ala Gly Thr Phe Val Lys Ala Pro Cys Glu Ile Pro His ThrGln Gly 65 70 75 80 Gln Cys Glu Lys Cys His Pro Gly Thr Phe Thr Glu LysAsp Asn Tyr 85 90 95 Leu Asp Ala Cys Ile Leu Cys Ser Thr Cys Asp Lys AspGln Glu Met 100 105 110 Val Ala Asp Cys Ser Ala Thr Ser Asp Arg Lys CysGln Cys Arg Thr 115 120 125 Gly Leu Tyr Tyr Tyr Asp Pro Lys Phe Pro GluSer Cys Arg Pro Cys 130 135 140 Thr Lys Cys Pro Gln Gly Ile Pro Val LeuGln Glu Cys Asn Ser Thr 145 150 155 160 Ala Asn Thr Val Cys Ser Ser SerVal Ser Asn Val Asp Thr His Thr 165 170 175 Cys Pro Pro Cys Pro Ala ProGlu Leu Leu Gly Gly Pro Ser Val Phe 180 185 190 Leu Phe Pro Pro Lys ProLys Asp Thr Leu Met Ile Ser Arg Thr Pro 195 200 205 Glu Val Thr Cys ValVal Val Asp Val Ser His Glu Asp Pro Glu Val 210 215 220 Lys Phe Asn TrpTyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 225 230 235 240 Lys ProArg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 245 250 255 LeuThr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 260 265 270Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 275 280285 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 290295 300 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val305 310 315 320 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn Gly 325 330 335 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp Ser Asp 340 345 350 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp 355 360 365 Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala Leu His 370 375 380 Asn His Tyr Thr Gln Lys Ser Leu Ser LeuSer Pro Gly Lys 385 390 395 15 11 PRT Artificial Sequence Description ofArtificial Sequence peptide 15 Tyr Gly Arg Lys Lys Arg Arg Gln Arg ArgArg 1 5 10

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of: (a) thenucleotide sequence as set forth in SEQ ID NOS: 7 or 9; (b) a nucleotidesequence encoding the polypeptide as set forth in SEQ ID NOS: 8 or 10;(c) a nucleotide sequence complementary to (a) or (b).
 2. A vectorcomprising the nucleic acid molecule of claim
 1. 3. A host cellcomprising the vector of claim
 2. 4. The host cell of claimed that is aeukaryotic cell.
 5. The host cell of claim 3 that is a prokaryotic cell.6. A process of producing a tmst2-receptor polypeptide comprisingculturing the host cell of claim 3 under suitable conditions to expressthe polypeptide, and optionally isolating the polypeptide from theculture.
 7. The process of claim 6 wherein the nucleic acid moleculecomprises promoter DNA other than the promoter DNA for the nativetmst2-receptor polypeptide operatively linked to the DNA encoding thetmst2-receptor polypeptide.
 8. A composition comprising a nucleic acidmolecule of claim 1 and a carrier.
 9. A composition of claim 8 whereinsaid nucleic acid molecule is contained in a viral vector.
 10. A viralvector comprising a nucleic acid molecule of claim
 1. 11. An isolatednucleic acid molecule comprising a nucleic acid sequence that is atleast 92% identical to the sequence of the nucleic acid molecule ofclaim 1 and encodes a polypeptide that is capable of binding TNF ligandTRAIL.
 12. An isolated nucleic acid molecule comprising a nucleic acidsequence that is at least 92% identical to the nucleic acid mnolecule ofclaim 1 and encodes a polypeptide that inhibits apoptosis.
 13. Anisolated nucleic acid molecule that encodes a polypeptide that iscapable of binding TNF ligand TRAIL and hybridizes to the complement ofthe nucleic acid molecule of claim 1 under the following stringentconditions: a final wash of 0.015 M sodium chloride and 0.0015 M sodiumcitrate at 65-68° C.
 14. An isolated nucleic acid molecule that encodesa polypeptide that inhibits apoptosis and hybridizes to the complementof the nucleic acid molecule of claim 1 under the following stringentconditions: a final wash of 0.015 M sodium chloride and 0.0015 M sodiumcitrate at 65-68° C.
 15. An isolated polynucleotide comprising a nucleicacid sequence that is at least 92% identical to the sequence of thenucleic acid molecule of claim 1 and encodes a polypeptide that is aTNF-receptor family nmber capable of binding TRAIL.
 16. An isolatednucleic acid molecule that encodes a polypeptide that is a TNF-receptorfamily member capable of binding TRAIL and hybridizes to the complementof the nucleic acid molecule of claim 1 under the following stringentconditions: a final wash of 0.015 M sodium chloride and 0.0015 M sodiumcitrate at 65-68° C.
 17. A fusion polypeptide comprising an amino acidsequence encoded by the nucleic acid sequence of any one of claims 1,11, 12, 13, 14, 15, and 16, fused to a heterologous amino acid sequence.18. The fusion polypeptide of claim 17 wherein the heterologous aminoacid sequence is an IgG constant domain or fragment thereof.
 19. Adiagnostic reagent comprising a nucleic acid molecule of any one ofclaims 1, 11, 12, 13, 14, 15, and 16, wherein the nucleic acid moleculeis detectably labeled.
 20. A diagnostic reagent comprising apolynucleotide that is detectably labeled and is complementary to anucleic acid molecule according to any one of claims 1, 11, 12, 13, 14,15, and 16.